Molecular Investigation of Francisella -Like Endosymbiont in Ticks and Francisella tularensis in Ixodid Ticks and Mosquitoes in Turkey

Abstract

This study was carried out to investigate the molecular prevalence of Francisella-like endosymbionts (FLEs) and Francisella tularensis in ticks (Acari: Ixodidae) and mosquitoes in Turkey. Genomic DNA pools were constructed from a total of 1477 adult hard ticks of Rhipicephalus (Rh.) annulatus, Rh. turanicus, Rh. sanguineus, Rh. bursa, Haemaphysalis (Hae.) parva, Hae. sulcata, Hyalomma marginatum marginatum, H. anatolicum anatolicum, H. anatolicum excavatum, H. detritum detritum, H. dromedarii, Dermacentor marginatus, and Ixodes ricinus species, which were collected from several barns, cattle, and people. Genomic DNA was also extracted from pools consisting of 6203 adult female mosquito species belonging to Aedes vexans, Culex (Cx.) pipiens, Cx. hortensis, Cx. theileri, Culiseta annulata, and Anopheles maculipennis species. Conventional PCR and TaqMan probe-based real- time PCR targeting the 16S rRNA gene for FLEs and the lpnA gene for F. tularensis, respectively, were performed on the DNA isolates obtained. FLEs and F. tularensis were not found in any genomic DNA pools constructed from ixodid ticks and mosquitos. This study represents the first investigation of F. tularensis and FLEs in potential vector ticks and mosquitoes by molecular methods in Turkey. The present study provides useful insights into the molecular epidemiology of F. tularensis and FLEs. One of the major conclusions of the study is that tularemia outbreaks may be essentially due to direct transmission from the environment (especially from water) in Turkey and not to vector-borne transmission.

Introduction

The Francisella genus within the rapidly expanding Francisellaceae family currently comprises the F. tularensis, F. novicida, F. philomiragia, F. piscicida, F. noatunensis, F. hispaniensis, F. halioticida, and F. guangzhouensis species, as well as the other nonpathogenic members Francisella-like endosymbionts (FLEs) (Birdsell et al. 2014, Szigeti et al. 2014). F. tularensis, a Gram-negative coccobacillus, which is regarded as a potential bioterrorism agent, causes the highly infectious zoonotic disease tularemia. F. tularensis subsp. tularensis (type A) and holarctica (type B) can sometimes be mortal in humans. F. tularensis subsp. mediasiatica is believed to be less virulent in humans than the other subspecies. F. tularensis subsp. holarctica is prevalent along the northern hemisphere whereas F. tularensis subsp. tularensis and mediasiatica are mostly seen in North America and Central Asia (Ellis et al. 2002, Sjöstedt 2005, Svensson et al. 2009, Mitchell et al. 2010, Carvalho et al. 2014).

People can acquire F. tularensis agents through contact with infected animals, consumption of contaminated food or water, inhalation of infectious aerosols, or the bites of infected arthropod vectors (Johansson et al. 2000a, Kugeler et al. 2005, Hazlett and Cirillo 2009, Petersen et al. 2009). Tularemia has been reported from many kinds of arthropod species, such as ticks, mosquitoes, flies, lice, fleas, midges, and bedbugs (Olsufiev 1943, Hopla 1974, Bell 1977, Petersen et al. 2009). Among these arthropods, only ixodid ticks, tabanids, and mosquitoes have been reported as the important vectors of F. tularensis in the transmission to people (Brown et al. 2005). Mosquitoes and tabanids are thought to be mechanical vectors of tularemia, whereas ticks are considered as the biological vectors (Philip and Parker 1932, Foil 1989, Petersen et al. 2009). In addition, ticks can maintain F. tularensis for a long time in the environment. Tick-borne transmission is generally seen in sporadic cases, but occasional outbreaks have been documented (Warring and Ruffin 1946, Saliba et al. 1966, Schmid et al. 1983, Markowitz et al. 1985). Epidemiological characteristics of vector-borne tularemia vary throughout the Northern Hemisphere. An arthropod bite is a common mode of transmission to humans in the United States, Sweden, Finland, and Russia, whereas arthropod-borne disease accounts for only a small percentage of human cases in Central Europe (Bell 1977, Hubalek et al. 1996, Tarnvik et al. 2004, Petersen et al. 2009).

On the basis of a high degree of similarity between 16S rRNA gene sequences, other microorganisms have been classified as probable members of the Francisellaceae family; these include FLEs (Kugeler et al. 2005, Petersen et al. 2009, Sjöstedt 2005, 2011, Carvalho et al. 2014). FLEs belong to a distinct phylogenetic clade from F. tularensis species (Escudero et al. 2008). FLEs have a worldwide distribution and are transmitted vertically by both hard and soft ticks (Sun et al. 2000, Escudero et al. 2008, Carvalho et al. 2011, Ivanov et al. 2011, Dergousoff and Chilton 2012). Studies on FLEs are hampered by their inability to grow on cell-free media; therefore, most of the molecular studies have been carried out with total DNA extracts from ticks rather than on FLE cultures (Ivanov et al. 2011). The first assays for the detection of tularemia based on conventional PCR amplification have led to the misidentification of FLEs and F. tularensis. However, new molecular methods (conventional PCR, nested PCR, real-time PCR, pulse-field gel electrophoresis [PFGE], amplified fragment-length polymorphism [AFLP], ribotyping, etc.) are more convenient for the differentiation of these species. On the other hand, the sequence analyses of the DNA products are essential for the confirmation of the PCR results (Kugeler et al. 2005).

FLEs have been detected in several countries by amplifying the sequences of the 16S rRNA and/or tul4 genes (Forsman et al. 1994, Johansson et al. 2000b, Kugeler et al. 2005, Zhang et al. 2008, Goethert and Telford 2009, Machado-Ferreira et al. 2009, Sreter-Lancz et al. 2009, Reye et al. 2010, Broman et al. 2011, Carvalho et al. 2011, Ivanov et al. 2011, Kreizinger et al. 2013). Several real-time PCR assays that appear to be more sensitive and specific than conventional PCR have been developed recently to determine F. tularensis (Wicki et al. 2000, Fujita et al. 2006, Versage et al. 2003, Tomaso et al. 2007, Svensson et al. 2009, Mitchell et al. 2010, Gehringer et al. 2013, Bonnet et al. 2013, Michelet et al. 2013, Toma et al. 2014). PCR assays targeting 16S rDNA (Forsman et al. 1994) or specific genes encoding outer membrane proteins, such as fopA (Fulop et al. 1996) and lpnA (Long et al. 1993, Junhui et al. 1996, Sjöstedt et al. 1997), have been used to detect Francisella.

The present study investigated the prevalence of FLEs and F. tularensis in some hard tick and mosquito populations from Turkey by conventional 16S rRNA-based PCR and real-time PCR.

Materials and Methods

Study areas

The study was conducted in the Kayseri region (38°56′0″N, 34°24′0″E), which is a large and industrialized city in Central Anatolia, Turkey. Tularemia outbreaks (at least 110 human cases) have been reported in Kayseri Province during periods in 2005–2013. Because the agents were isolated from several water sources in the area, water-borne transmission was suggested for all human cases that were clinically characterized as being oropharyngeal and glandular forms (Balci et al. 2014).

Ticks, mosquitoes, and the construction of genomic DNA pools

Adult tick and mosquito specimens used in the study were obtained from several projects (The Scientific and Technical Research Council of Turkey with code numbers 107O533 and 113O202; Erciyes University Research Fund with project code VA-05-05) previously carried out in the research area during different time periods from 2006 to 2013. A total of 1198 pools (599 head-thorax + 599 abdomen) (1–17 samples/pool, the pools consisted of the same mosquito species) assembled from 6203 adult female mosquito species (3202 Aedes [Ae.] vexans, 2610 Culex [Cx.] pipiens, 36 Cx. hortensis, 193 Cx. theileri, 100 Culiseta annulata, 62 Anopheles maculipennis) and 38 pools (1–96 samples/pool) were used. The ticks were grouped (38 groups) according to the sampling locations and species, and each group consisted of same tick species. If there were more than 10 ticks in each group, these groups were subdivided (e.g., 96 Haemaphysalis (Hae.) parva ticks = 10 subgroups).

Following the DNA extractions from the whole tick bodies, the DNA elutions belonging to these subgroups were combined in one eppendorf tube, and these tubes were called one pool (96 Hae. parva ticks in one DNA pool) assembled from 1115 adult ixodid ticks (267 Rhipicephalus (Rh.) annulatus, 106 Hae. parva, 2 Hae. sulcata, 245 Hyalomma marginatum marginatum, 106 Hyalomma anatolicum anatolicum, 53 H. anatolicum excavatum, 68 H. detritum detritum, 219 Rh. turanicus, 28 Rh. bursa, 1 Rh. sanguineus, 20 Dermacentor marginatus) that were collected from infested cattle and several barns in Kayseri region were constructed. In addition, a further 42 pools (3–10 samples/pool, the pools consisted of the same tick species [whole tick bodies]) were also constructed from 362 adult ixodid ticks (39 Hae. parva, 3 Hae. sulcata, 86 H. m. marginatum, 14 H. a. anatolicum, 20 Hyalomma a. excavatum, 3 H. d. detritum, 13 H. dromedarii, 114 Rh. turanicus, 38 Rh. bursa, 16 Rh. sanguineus, 11 D. marginatus, and 5 Ixodes ricinus) collected from infested people in the same region.

Genomic DNA isolation

Genomic DNA was extracted from pools by using a Multisource Genomic DNA Kit (AP-MN-MS-GDNA-250, Axygen Biosciences, Union City, CA) according to the manufacturer's instructions and eluted in 50 μL of elution buffer. The extracted DNAs were stored at −20°C until the analyses. The DNA concentrations of the samples were measured in a NanoDrop Spectrophotometer (cat. no. ASP-3700, ACT Gene, Piscataway, NJ) to adjust the optimum amount of DNA used in the PCR mastermix.

TaqMan real-time PCR analyses

Screening of F. tularensis in the tick and mosquito DNA pools was performed using a IpnA gene-specific TaqMan probe–based real-time PCR assay in Stratagene Mx 3005P (Stratagene, Agilent Technologies, Santa Clara, CA). Each reaction consisted of 50 ng of template DNA, 2× TaqMan Universal PCR Mastermix, 20 mM each of the primers iQFt1F (5′-CGCAGGTTTAGCGAGCTGTT-3′) and iQFt1R (5′-GCAGCTTGCTCAGTAGTAGCT GTCT-3′), 5 μM iQFt1 Probe (5′-FAM-CATCATCAGAGCCACCTA ACCCTA-3′), and sterile deionized water to produce a total volume of 25 μL. PCR conditions were initial denaturation at 50°C for 2 min and 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s (Thelaus et al. 2009). All samples were analyzed in duplicate for qPCR reactions. The F. tularensis subsp. holarctica (NCTC 10857) strain obtained from Public Health Agency of Turkey and sterile deionized water were used as positive and negative controls in all reactions.

16S rRNA PCR analyses

Amplification of the 16S rRNA gene region was also performed on the tick DNA pools to detect FLEs. Each reaction consisted of 50 ng of template DNA, 2.5 μL of 10× PCR buffer, 4 mM MgCl₂, 0.4 μM each Francisella-specific Fr153F0.1 (5′-GCCCATTTGAGGGGGATACC-3′) and Fr1281R0.1 (5′-GGACTAAGAG TACCTTTTTGAGT-3′) primers, 200 mM each deoxynucleotide (dNTP), 1 U of Taq DNA polymerase, and sterile deionized water to produce a total volume of 25 μL. PCR conditions were initial denaturation at 94°C for 4 min, 40 cycles of 94°C for 30 s, 60°C for 45 s, and 72°C for 60 s, and a final incubation at 72°C for 20 min in a thermal cycler (modified from Broman et al. 2011). Positive and negative controls were used as described in a real-time PCR assay.

Results

TaqMan real-time PCR analyses

According to the real-time PCR analyses, no F. tularensis positivity was detected in any genomic DNA pools constructed from the 1477 adult ixodid ticks collected from cattle, barns, and humans nor in the 6203 adult mosquitos (Table 1).

Table 1.

The Examined Tick and Mosquito Species for the Presence of F. tularensis and FLEs

		Number of samples
	Mosquito/tick species	n	%	F. tularensis positives	FLEs positives
Mosquito species	Aedes vexans	3202	51.62	0
	Culex pipiens	2610	42.08	0
	Culex theileri	193	3.11	0
	Culex hortensis	36	0.58	0
	Culiseta annulata	100	1.61	0
	Anopheles maculipennis	62	1.00	0
	Total	6203	100.00	0
Tick species	Rhipicephalus annulatus	267	18.08	0	0
	Rhipicephalus turanicus	333	22.55	0	0
	Rhipicephalus bursa	66	4.47	0	0
	Rhipicephalus sanguineus	17	1.15	0	0
	Haemaphysalis parva	145	9.82	0	0
	Haemaphysalis sulcata	5	0.34	0	0
	Hyalomma marginatum	331		0	0
	marginatum		22.41
	Hyalomma anatolicum	120		0	0
	anatolicum		8.12
	Hyalomma anatolicum	73		0	0
	excavatum		4.94
	Hyalomma detritum detritum	71	4.81	0	0
	Hyalomma dromedarii	13	0.88	0	0
	Dermacentor marginatus	31	2.10	0	0
	Ixodes ricinus	5	0.34	0	0
	Total	1477	100.00	0	0

FLEs, Francisella-like endosymbionts.

Amplification of 16S rRNA

According to the results of conventional PCR analyses, no positive DNA amplifications were determined in any genomic DNA pools from adult ticks.

Discussion

Tularemia is an endemic disease in Turkey. The first tularemia epidemic in Turkey was reported in 1936 in the Thrace region. In the years following this outbreak, many epidemic or sporadic cases were reported from different regions in Turkey (Akalin et al. 2009). After two consecutive outbreaks in 1945 in the Thrace region and in 1953 in southern Turkey, there was a sharp decline in the number of cases. A total of 507 tularemia cases were described in Turkey during 1988–2004. Tularemia was placed in the list of nationally notifiable diseases in 2005, and from this time to 2011, approximately 4824 cases were reported from all of Turkey (Kilic 2010, Ulu Kilic et al. 2013, Mengeloglu et al. 2014, Balci et al. 2014). Many experts suggested that the outbreaks in Turkey are generally related to water, and infection was only observed in rural areas, particularly among farming families, including in housewives, children, hunters, and forest workers. In these areas, contaminated water and/or food sources could be attributed to F. tularensis infection (Balci et al. 2014, Mengeloglu et al. 2014). The first case of tularemia was recorded in Kayseri Province in 2005. Then, two cases were reported in 2006 and one case in 2007. There were no cases between 2007 and 2009, which was followed by an outbreak in 2010. During 2010, 21 cases were recorded in seven towns, 62 cases in 2011, and 27 cases in 2012. A total of 110 cases were recorded in 12 out of 16 towns in Kayseri Province between 2010 and 2012 (Balci et al. 2014).

Despite many cases reported per year in Turkey, there has been no study investigating F. tularensis in possible arthropod vectors. There are two studies on the presence of tularemia in animals, one report from a wild rodent (Celebi et al. 2012) and one report from two mice in Thrace region (Unal Yilmaz et al. 2014). In addition there are two GenBank records, a water-borne F. tularensis subsp. holarctica isolate from Gudul, Ankara (GenBank acc. no. JX436321) and a water-borne F. tularensis subsp. holarctica biovar japonica isolate (GenBank acc. no. CP007148). Due to the previously reported human cases in the Kayseri region, this study was constructed on the possible role of hard ticks and mosquito species in the transmission of tularemia in the region.

In the previous studies (Philip and Parker 1932), the adult mosquitos and deer flies were suggested as the mechanical vector of tularemia, but which Francisella subspecies caused the infection was not known in these studies. In recent years, clinical and epidemiological studies suggested that the mosquito-borne transmission is one of the most significant transmission ways of tularemia (Gurycova 1998, Eliasson et al. 2002, Hanke et al. 2009, Triebenbach et al. 2010, Mahajan et al. 2011, Lundstrom et al. 2011, Ryden et al. 2012, Thelaus et al. 2014). Lundstrom et al. (2011) detected F. tularensis subsp. holarctica in adult Ae. punctor, Ae. sticticus, and Ae. vexans based on the lpnA gene by real-time PCR in the tularemia endemic areas of Sweden.

Triebenbach et al. (2010) determined F. tularensis DNA in adult Ochlerotatus communis, O. fitchii, O. excrucians, O. pionips, Cs. alaskaensis, Cs. impatiens, Cs. incidens, and Ae. vexans for the Francisella fopA gene via real-time qPCR in Alaska. Carvalho et al. (2012) investigated a total of 4949 mosquitoes of Culex spp., Ochlerotatus spp., Anopheles spp., Culiseta spp., and Ae. aegypti by nested PCR analyses for the tul4 gene region of F. tularensis in Portugal, and they found that all samples were negative for the genomic DNA presence of F. tularensis. Thelaus et al. (2014) speculated that transmission of tularaemia by mosquitoes in nature occurs at much lower frequencies than could be detected in their study. In addition, they reported that F. tularensis subsp. holarctica was found in several genera and species of wild-caught mosquitoes in Sweden.

In our study, we investigated a total of 6203 adult mosquitos of Ae. vexans, Cx. pipiens, Cx. hortensis, Cx. theileri, C. annulata, and A. maculipennis for the F. tularensis IpnA gene by real-time PCR in the Kayseri region. We found that all samples were negative for F. tularensis, similar to the study results of Carvalho et al. (2012). Recent investigations performed with molecular methods have shown that various mosquito species exposed to F. tularensis subsp. holarctica during their aquatic larval stage maintain the bacterium or its DNA until the adult stage (Lundstrom et al. 2011, Thelaus et al. 2014, Backman et al. 2015). The tularemia-positive and -negative findings in different mosquito species may arise from the differences in the ecological parameters for vectors and the reservoirs of Francisella species.

Our findings do not suggest natural transmission of F. tularensis from its water reservoir via female mosquitoes to their vertebrate hosts, including humans in the tularemia-endemic Kayseri region of Central Anatolia. In addition, our results suggest that mosquitoes did not come in contact with the tularemia agent F. tularensis in the natural aquatic environments in the region. However, further studies are needed to confirm the possible roles of mosquitos in tularemia-endemic regions and to investigate any potential relation between F. tularensis and a specific mosquito species.

Recent molecular studies have shown the presence of FLEs, which are closely related to F. tularensis (Noda et al. 1997) in both hard and soft ticks (Escudero et al. 2008, Carvalho et al. 2011, Ivanov et al. 2011, Sun et al. 2000, Dergousoff and Chilton 2012). The discrimination between F. tularensis and FLEs is significantly critical for preventing misidentifications in epidemiological studies for tularemia. In recent years, various molecular studies by conventional PCR, real-time PCR, and sequencing analyses based on different gene regions of FLEs and F. tularensis have been performed in the world. In Germany, Gehringer et al. (2013) investigated adult I. ricinus and D. marginatus ticks by real-time PCR of the 16S rRNA gene region, and they found that all Francisella-positive I. ricinus samples clustered with F. tularensis sequences, whereas all Dermacentor tick samples clustered with FLEs sequences in the phylogenetic analyses. Similarly, Wicki et al. (2000) reported a 0.12% F. tularensis positivity in I. ricinus ticks by TaqMan real-time PCR in Switzerland.

In France, Bonnet et al. (2013) analyzed D. marginatus, D. reticulatus, and I. ricinus ticks for tularemia by real-time PCR, and no F. tularensis positivity was found in any samples whereas F. philomiragia was detected in D. reticulatus (18.9%) and D. marginatus (1.3%) samples. In a similar study conducted in Italy, Toma et al. (2014) found no F. tularensis positivity in H. m. marginatum, H. m. rufipes, Hyalomma spp., Amblyomma spp., I. ricinus, and Ixodes spp. collected from migratory birds by real-time PCR. Zhang et al. (2008) detected 1.98% positivity for F. tularensis in D. silvarum and I. persulatus ticks by nested PCR in tularemia endemic regions of China and confirmed the positives as F. tularensis subsp. holarctica by sequence and cluster analyses.

In Brazil, Machado-Ferreira et al. (2009) detected Francisella positivity in one each samples of Amblyomma dubitatum, D. nitens, and Rh. microplus samples by PCR for 16S rDNA and tul4 genes and confirmed all three positive samples as FLEs in sequence analyses. Similarly, Kugeler et al. (2005) determined Francisella DNA in two of D. variabilis and 14 of D. occidentalis pools by PCR. According to the sequence analyses of 16S rRNA gene region, they found all positive samples to be FLEs. In Bulgaria, Ivanov et al. (2011) analyzed Rh. sanguineus, D. marginatus, I. ricinus, H. m. marginatum, D. reticulatus, Rh. bursa, Rh. turanicus, and H. aegyptium species for the presence of Francisella spp. by 16S rRNA PCR and detected FLEs in H. m. marginatum, H. aegyptium, Rh. sanguineus, and D. reticulatus species. In Portugal, Carvalho et al. (2011) identified FLEs in D. reticulatus ticks (39.0%) among D. reticulatus, I. hexagonus, I. ricinus, I. frontalis, and D. marginatus species.

Goethert and Telford (2009) reported a 3.4% annual prevalence of F. tularensis in Dermacentor variabilis ticks by PCR in the United States. In Luxembourg, Reye et al. (2010) reported no F. tularensis positivity in adult I. ricinus ticks. In a similar study, Sreter-Lancz et al. (2009) investigated D. reticulatus, 211 I. ricinus, and Hae. concinna adults from Hungary for the presence of F. tularensis by PCR assays and determined no Francisella-specific amplification products in I. ricinus and Hae. concinna samples. However they identified FLEs in D. reticulatus (1.2%) in the phylogenetic analyses.

In another study from Hungary, Kreizinger et al. (2013) investigated I. ricinus, I. acuminatus, D. marginatus, D. reticulatus, Hae. inermis, Hae. concinna, and Hae. punctata ticks by PCR targeting the 16S rRNA and tul4 genes. They detected F. tularensis subsp. holarctica in two pools of Hae. concinna and one pool of D. reticulatus, both representing a minimum prevalence of 0.27%. The sequences of a FLE were detected in 11 pools of D. reticulatus showing a minimum prevalence of 3.0%.

In our study, we investigated adult D. marginatus, I. ricinus, Rh. annulatus, Rh. turanicus, Rh. bursa, Rh. sanguineus, Hae. parva, Hae. sulcata, H. m. marginatum, H. a. anatolicum, H. a. excavatum, H. detritum, and H. dromedarii ticks from barns, cattle, and people by TaqMan probe based real-time PCR for the presence of F. tularensis, and by 16S rRNA conventional PCR for the presence of FLEs. We did not find any positivity for F. tularensis and FLEs in any tick samples, a result similar to some previous studies (Reye et al. 2010, Bonnet et al. 2013, Toma et al. 2014). In a general overview of tick-borne tularemia studies in the world, it has been considered that F. tularensis could be found in all ixodid tick species, with higher prevalence in Ixodes and Dermacentor species. However, the isolates from Dermacentor species were generally found closer to FLEs than to F. tularensis in the phylogenetic analyses.

In our study, we used a real-time PCR probe-based assay, which can provide high specificity due to binding of two primers to a probe, for detection of the F. tularensis-specific lpnA sequence, as previously described (Thelaus et al. 2009). The lpnA assay generates a product from all four F. tularensis subspecies, but not from other Francisella spp. or FLEs. The detection limit of the lpnA assay was estimated as 10³ bacteria/mL in natural water samples (Thelaus et al. 2009, Broman et al. 2011). In addition, Thelaus et al. (2014) reported that real-time PCR detection of the F. tularensis lpnA gene was possible for concentrations between 10³ and 10⁶ bacteria per mosquito. Broman et al. (2011) have reported that the lpnA assay is potentially capable of detecting both pathogenic F. tularensis (i.e., F. tularensis subsp. tularensis and holarctica) and nonpathogenic F. tularensis.

Specific discrimination between F. tularensis and FLEs is definitely an essential point in the epidemiological studies on tularemia. To discriminate F. tularensis and FLEs in lpnA assays, 16S rRNA PCR and sequence analyses are required to determine FLEs in complex samples. For this perspective, Michelet et al. (2013) performed four different available molecular detection techniques on D. reticulatus ticks—16S rDNA (Forsman et al. 1994), tul4 (Long et al. 1993), and tul4 and fopA gene PCR amplifications using real-time TaqMan PCR assays (Versage et al. 2003). According to their results, they found that tul4 and fopA real-time PCR assays can easily and effectively discriminate between F. tularensis and FLEs in D. reticulatus. Similarly, Versage et al. (2003) developed a multitarget real-time TaqMan PCR assay for enhanced detection of F. tularensis in complex specimens. They reported that the combined use of the multitarget TaqMan assay (ISFtu2, 23kDa, and tul4) was highly specific, displaying no cross-reactivity with the non-Francisella bacteria tested and capable of differentially diagnosing both F. tularensis and F. philomiragia.

Conclusions

This study represents the first molecular epidemiological approach to determine mosquitoes and ticks as potential vectors of F. tularensis and FLEs in Turkey. Because no bacterial DNA could be detected in any of the tested samples, one of the major conclusions of the study is that tularemia outbreaks may be essentially due to direct transmission from the environment (especially from water) in Turkey and not to vector-borne transmission. However, it could not be considered that the ticks and mosquitoes do not play a significant role in the transmission of tularemia, and further studies are needed to explore possible vector-borne tularemia in Turkey and to validate such an hypothesis.

Footnotes

Acknowledgments

This study was financially supported by The Scientific and Technical Research Council of Turkey (TÜBİTAK, project no. 113O202) and Erciyes University Research Fund (project no. TDA-2014-5172). The authors thank Public Health Agency of Turkey for procurement of the positive genomic DNAs.

Author Disclosure Statement

No competing financial interests exist.

References

Akalin

, Helvaci

, Gedikoglu

. Re-emergence of tularemia in Turkey. Int J Infect Dis, 2009; 13:547–551.

Backman

, Naslund

, Forsman

, Thelaus

. Transmission of tularemia from a water source by transstadial maintenance in a mosquito vector. Sci Rep, 2015; 5:7793.

Balci

, Borlu

, Kilic

, Demiraslan

, et al. Tularemia outbreaks in Kayseri, Turkey: An evaluation of the effect of climate change and climate variability on tularemia outbreaks. J Infect Public Health, 2014; 7:125–132.

Barns

, Grow

, Okinaka

, Keim

, et al. Detection of diverse new Francisella-like bacteria in environmental samples. Appl Environ Microbiol, 2005; 71:5494–5500.

Bell

. Tularemia. CRC Handbook Series in Zoonoses, Section A. Boca Raton, FL: CRC Press, 1977:161–193.

Birdsell

, Vogler

, Buchhagen

, Clare

, et al. TaqMan real-time PCR assays for single-nucleotide polymorphisms which identify Francisella tularensis and its subspecies and subpopulations. PLoS One, 2014; 9:e107964.

Bonnet

, de la Fuente

, Nicollet

, Liu

, et al. Prevalence of tick-borne pathogens in adult Dermacentor spp. ticks from nine collection sites in France. Vector Borne Zoonotic Dis, 2013; 13:226–236.

Broman

, Thelaus

, Andersson

, Backman

, et al. Molecular detection of persistent Francisella tularensis subspecies holarctica in natural waters. Int J Microbiol, 2011; 851946.

Brown

, Lane

, Dennis

. Geographic distribution of tick-borne diseases and their vectors. In: Goodman

, Dennis

, Sonenshine

, eds. Tick-Borne Diseases of Humans. Washington, DC: ASM Press, 2005:363–391.

10.

Carvalho

, Santos

, Soares

, Ze-Ze

, et al. Francisella-like endosymbiont in Dermacentor reticulatus collected in Portugal. Vector Borne Zoonotic Dis, 2011; 11:185–188.

11.

Carvalho

, Ze-Ze

, Duarte

, Nuncio

, et al. Screening of mosquitoes as vectors of Francisella tularensis in Portugal. 7th International Conference on Tularemia, Sept 17–20, 2012, Breckenridge, Colorado.

12.

Carvalho

, Lopes de Carvalho

, Ze-Ze

, et al. Tularaemia: A challenging zoonosis. Comp Immunol Microbiol Infect Dis, 2014; 37:85–96.

13.

Celebi

, Kilic

, Karagoz

, Durmaz

. Ulkemizde izole edilen Francisella tularensis suslarinin alt turlerinin belirlenmesi. X. Ulusal Veteriner Hekimleri Mikrobiyoloji Kongresi (Uluslararasi Katilimli), 24–27 Eylul, Kusadasi/Aydin, Turkiye, 2012:36–37.

14.

Dergousoff

, Chilton

. Association of different genetic types of Francisella-like organisms with the rocky mountain wood tick (Dermacentor andersoni) and the American dog tick (Dermacentor variabilis) in localities near their northern distributional limits. Appl Environ Microbiol, 2012; 78:965–971.

15.

Eliasson

, Lindback

, Nuorti

, Arneborn

, et al. The 2000 tularemia outbreak: A case-control study of risk factors in disease-endemic and emergent areas, Sweden. Emerg Infect Dis, 2002; 8:956–960.

16.

Ellis

, Oyston

PCF

, Green

, Titball

. Tularemia. Clin Microbiol Rev, 2002; 15:629–646.

17.

Escudero

, Toledo

, Gil

, Kovacsova

, et al. Molecular method for discrimination between Francisella tularensis and Francisella-like endosymbionts. J Clin Microbiol, 2008; 46:3139–3143.

18.

Foil

. Tabanids as vectors of disease agents. Parasitol Today, 1989; 5:88–96.

19.

Forsman

, Sandström

, Sjöstedt

. Analysis of 16S ribosomal DNA sequences of Francisella strains and utilization for determination of the phylogeny of the genus and for identification of strains by PCR. Int J Syst Bacteriol, 1994; 44:38–46.

20.

Fujita

, Tatsumi

, Tanabayashi

, Yamada

. Development of a real-time PCR assay for detection and quantification of Francisella tularensis . J Infect Dis, 2006; 59:46–51.

21.

Fulop

, Leslie

, Titball

. A rapid, highly sensitive method for the detection of Francisella tularensis in clinical samples using the polymerase chain reaction. Am J Trop Med Hyg, 1996; 54:364–366.

22.

Gehringer

, Schacht

, Maylaender

, Zeman

, et al. Presence of an emerging subclone of Francisella tularensis holarctica in Ixodes ricinus ticks from south-western Germany. Ticks Tick Borne Dis, 2013; 4:93–100.

23.

Goethert

, Telford

. Nonrandom distribution of vector ticks (Dermacentor variabilis) infected by Francisella tularensis . PLoS Pathog, 2009; 5:e1000319.

24.

Gurycova

. First isolation of Francisella tularensis subsp. tularensis in Europe. Eur J Epidemiol, 1998; 14:797–802.

25.

Hanke

, Otten

, Berner

, Serr

, et al. Ulceroglandular tularemia in a toddler in Germany after a mosquito bite. Eur J Pediatr, 2009; 168:937–940.

26.

Hazlett

, Cirillo

. Environmental adaptation of Francisella tularensis . Microbes Infect, 2009; 11:828–834.

27.

Hopla

. The ecology of tularemia. Adv Vet Sci Comp Med, 1974; 18:25–53.

28.

Hubalek

, Treml

, Halouzka

, Juricova

, et al. Frequent isolation of Francisella tularensis from Dermacentor reticulatis ticks in an enzootic focus of tularaemia. Med Vet Entomol, 1996; 10:241–246.

29.

Ivanov

, Mitkova

, Reye

, Hubschen

, et al. Detection of new Francisella-like tick endosymbionts in Hyalomma spp. and Rhipicephalus spp. (Acari: Ixodidae) from Bulgaria. Appl Environ Microbiol, 2011; 77:5562–5565.

30.

Johansson

, Berglund

, Eriksson

, Goransson

, et al. Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia. J Clin Microbiol, 2000a; 38:22–26.

31.

Johansson

, Ibrahim

, Goransson

, Eriksson

, et al. Evaluation of PCR-based methods for discrimination of Francisella species and subspecies and development of a specific PCR that distinguishes the two major subspecies of Francisella tularensis . J Clin Microbiol, 2000b; 38:4180–4185.

32.

Junhui

, Ruifu

, Jianchun

, Songle

, et al. Detection of Francisella tularensis by the polymerase chain reaction. J Med Microbiol, 1996; 45:477–482.

33.

Kilic

. A general overview of Francisella tularensis and the epidemiology of tularemia in Turkey. Flora, 2010; 15:37–58.

34.

Kreizinger

, Hornok

, Dan

, Hresko

, et al. Prevalence of Francisella tularensis and Francisella-like endosymbionts in the tick population of Hungary and the genetic variability of Francisella-like agents. Vector Borne Zoonotic Dis, 2013; 13:160–163.

35.

Kugeler

, Gurfield

, Creek

, Mahoney

, et al. Discrimination between Francisella tularensis and Francisella-like endosymbionts when screening ticks by PCR. Appl Environ Microbiol, 2005; 71:7594–7597.

36.

Long

, Oprandy

, Narayanan

, Fortier

, et al. Detection of Francisella tularensis in blood by polymerase chain reaction. J Clin Microbiol, 1993; 31:152–154.

37.

Lundstrom

, Andersson

, Backman

, Schafer

, et al. Transstadial transmission of Francisella tularensis holarctica in mosquitoes, Sweden. Emerg Infect Dis, 2011; 17:794–799.

38.

Machado-Ferreira

, Piesman

, Zeidner

, Soares

. Francisella-like endosymbiont DNA and Francisella tularensis virulence-related genes in Brazilian ticks (Acari: Ixodidae). J Med Entomol, 2009; 46:369–374.

39.

Mahajan

, Gravgaard

, Turnbull

, Jacobs

, et al. Larval exposure to Francisella tularensis LVS affects fitness of the mosquito Culex quinquefasciatus . FEMS Microbiol Ecol, 2011; 78:520–530.

40.

Markowitz

, Hynes

, de la Cruz

, Campos

, et al. Tick-borne tularemia. An outbreak of lymphadenopathy in children. JAMA, 1985; 254:2922–2925.

41.

Mengeloglu

, Duran

, Hakyemez

, Ocak

, et al. Evaluation of patients with tularemia in Bolu province in northwestern Anatolia, Turkey. J Infect Dev Ctries, 2014; 8:315–319.

42.

Michelet

, Bonnet

, Madani

, Moutailler

. Discriminating Francisella tularensis and Francisella-like endosymbionts in Dermacentor reticulatus ticks: evaluation of current molecular techniques. Vet Microbiol, 2013; 163:399–403.

43.

Mitchell

, Chatwell

, Christensen

, Diaper

, et al. Development of real-time PCR assays for the specific detection of Francisella tularensis ssp. tularensis, holarctica and mediasiatica . Mol Cell Probes, 2010; 24:72–76.

44.

Noda

, Munderloh

, Kurtti

. Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals. Appl Environ Microbiol, 1997; 63:3926–3932.

45.

Olsufiev

. Parasitology of tularemia. In: Khatenever

, ed. Tularemia Infection. Moscow, Russia; 1943:74–92.

46.

Petersen

, Mead

, Schriefer

. Francisella tularensis: An arthropod-borne pathogen. Vet Res, 2009; 40:7.

47.

Philip

, Parker

. Experimental transmission of tularaemia by mosquitoes. Public Health Rep, 1932; 47:2077–2088.

48.

Reye

, Hubschen

, Sausy

, Muller

. Prevalence and seasonality of tick-borne pathogens in questing Ixodes ricinus ticks from Luxembourg. Appl Environ Microbiol, 2010; 76:2923–2931.

49.

Ryden

, Bjork

, Schafer

, Lundstrom

, et al. Outbreaks of tularemia in a boreal forest region depends on mosquito prevalence. J Infect Dis, 2012; 205:297–304.

50.

Saliba

, Harmston

, Diamond

, Zymet

, et al. An outbreak of human tularemia associated with the American dog tick, Dermacentor variabilis . Am J Trop Med Hyg, 1966; 15:531–538.

51.

Schmid

, Kornblatt

, Connors

, Patton

, et al. Clinically mild tularemia associated with tick-borne Francisella tularensis . J Infect Dis, 1983; 148:63–67.

52.

Sjöstedt

, Eriksson

, Berglund

, Tarnvik

. Detection of Francisella tularensis in ulcers of patients with tularemia by PCR. J Clin Microbiol, 1997; 35:1045–1048.

53.

Sjöstedt

. Francisella. In: Brenner

, Krieg

, Staley

, Garrity

, eds. The Proteobacteria. New York: Springer-Verlag, 2005:200–210.

54.

Sjöstedt

. Special topic on Francisella tularensis and tularemia. Front Microbiol, 2011; 2:86.

55.

Sreter-Lancz

, Szell

, Sreter

, Marialigeti

. Detection of a novel Francisella in Dermacentor reticulatus: A need for careful evaluation of PCR-based identification of Francisella tularensis in Eurasian ticks. Vector Borne Zoonotic Dis, 2009; 9:123–126.

56.

Sun

, Scoles

, Fish

, O'Neill

. Francisella-like endosymbionts of ticks. J Invertebr Pathol, 2000; 76:301–303.

57.

Svensson

, Granberg

, Karlsson

, Neubauerova

, et al. A real-time PCR array for hierarchical identification of Francisella isolates. PLoS One, 2009; 4:e8360.

58.

Szigeti

, Kreizinger

, Hornok

, Abichu

, et al. Detection of Francisella-like endosymbiont in Hyalomma rufipes from Ethiopia. Ticks Tick Borne Dis, 2014; 5:818–820.

59.

Tarnvik

, Priebe

, Grunow

. Tularaemia in Europe: An epidemiological overview. Scand J Infect Dis, 2004; 36:350–355.

60.

Thelaus

, Andersson

, Mathisen

, Forslund

, et al. Influence of nutrient status and grazing pressure on the fate of Francisella tularensis in lake water. FEMS Microbiol Ecol, 2009; 67:69–80.

61.

Thelaus

, Andersson

, Broman

, Backman

, et al. Francisella tularensis subspecies holarctica occurs in Swedish mosquitoes, persists through the developmental stages of laboratory-infected mosquitoes and is transmissible during blood feeding. Microb Ecol, 2014; 67:96–107.

62.

Toma

, Mancini

, Di Luca

, Cecere

, et al. Detection of microbial agents in ticks collected from migratory birds in central Italy. Vector Borne Zoonotic Dis, 2014; 14:199–205.

63.

Tomaso

, Scholz

, Neubauer

, Al Dahouk

, et al. Real time PCR using hybridization probes for the rapid and specific identification of Francisella tularensis subspecies tularensis . Mol Cell Probes, 2007; 21:12–16.

64.

Triebenbach

, Vogl

, Lotspeich-Cole

, Sikes

, et al. Detection of Francisella tularensis in Alaskan mosquitoes (Diptera: Culicidae) and assessment of a laboratory model for transmission. J Med Entomol, 2010; 47:639–648.

65.

Ulu-Kilic

, Gulen

, Sezen

, Kilic

, et al. Tularemia in Central Anatolia. Infection, 2013; 41:391–399.

66.

Unal Yilmaz

, Gurcan

, Ozkan

, Karadenizli

. Trakya bölgesi'nde farelerde kültür, seroloji ve moleküler yöntemlerle Francisella tularensis varliginin aranmasi. Mikrobiyol Bul, 2014; 48:213–222.

67.

Versage

, Severin

DDM

, Chu

, Petersen

. Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens. J Clin Microbiol, 2003; 41:5492–5499.

68.

Warring

, Ruffin

. A tick-borne epidemic of tularemia. N Engl J Med, 1946; 234:137–140.

69.

Wicki

, Sauter

, Mettler

, Natsch

, et al. Swiss Army Survey in Switzerland to determine the prevalence of Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and tick-borne encephalitis virus in ticks. Eur J Clin Microbiol Infect Dis, 2000; 19:427–432.

70.

Zhang

, Liu

, Wu

, Xin

, et al. Detection of Francisella tularensis in ticks and identification of their genotypes using multiple-locus variable-number tandem repeat analysis. BMC Microbiol, 2008; 8:152.