Exp Appl Acarol (2014) 63:579–585 DOI 10.1007/s10493-014-9792-0
Rickettsia raoultii, the predominant Rickettsia found in Dermacentor silvarum ticks in China–Russia border areas Jing Wen • Dan Jiao • Jian-hua Wang • De-hai Yao • Zhi-xiang Liu Gang Zhao • Wen-dong Ju • Cheng Cheng • Yi-jing Li • Yi Sun
•
Received: 5 November 2013 / Accepted: 5 March 2014 / Published online: 4 April 2014 Ó Springer International Publishing Switzerland 2014
Abstract Since the year 2000, clinical patterns resembling tick-borne rickettsioses have been noticed in China–Russia border areas. Epidemiological data regarding species of the aetiological agent, tick vector prevalence and distribution as well as incidence of human cases in the areas are still sparse to date. In order to identify Rickettsia species occurring in the areas, we investigated Dermacentor silvarum collected in the selected areas. Rickettsia raoultii was the predominant Rickettsia found in D. silvarum evident with ompA, ompB, gltA and 17 kDa protein genes. The Rickettsia prevalence in D. silvarum appeared to be 32.25 % with no sex difference. The results extend the common knowledge about the geographic distribution of R. raoultii and its candidate vector tick species, which suggest an emerged potential threat of human health in the areas. Keywords
R. raoultii D. silvarum China–Russia border area
J. Wen Y. Li (&) College of Veterinary Medicine, Northeast Agricultural University, Harbin City, Heilongjiang Province, China e-mail: [email protected] J. Wen e-mail: [email protected]; [email protected] J. Wen D. Jiao J. Wang D. Yao Z. Liu G. Zhao Heihe Entry-Exit Inspection and Quarantine Bureau, Heihe City, Heilongjiang Province, China e-mail: [email protected] G. Zhao e-mail: [email protected] W. Ju C. Cheng Heilongjiang Entry-Exit Inspection and Quarantine Bureau, Harbin City, Heilongjiang Province, China Y. Sun (&) Department of Vector Biology and Control, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China e-mail: [email protected]; [email protected]
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Introduction Rickettsia are obligate intracellular gram-negative bacteria which belong to the Spotted Fever Group (SFG) Rickettsiae. Rickettsia are associated with arthropod vectors and cause mild to fatal diseases in human (Fournier and Raoult 2009). In 1999, Rickettsia raoultii was found from Dermacentor nutallii in Siberia and Rhipicephalus pumilio in the Astrakhan region of former Soviet Union accoding to phylogenetic analysis of rrs (16s rDNA), Citrate synthase (gltA) and Outer membrane B (ompB) (Rydkina et al. 1999). In 2002, R. raoultii DNA was detected in a D. marginatus tick taken from the scalp of a patient in whom Tick-borne lymphadenopathy (TIBOLA) or Dermacentor-borne necrosis erythema lymphadenopathy (DEBONEL) developed in France (Mediannikov et al. 2008). And soon R. raoultii was recognized as one of caused agents of TIBOLA/DEBONEL (Parola et al. 2009), which is nowadays the most prevalent tick-borne rickettsiose in Europe Oteo and Portillo (2012). In China–Russia border areas, human cases of tick-borne rickettsioses have been increasing in recent years. From 2000 to 2012, over 500 cases were reported to National Center of Disease prevention and Control of China, most cases occur in northeast China, mainly in the China–Russia border areas. Most of the cases were diagnosed by clinical symptoms, and in some cases confirmed by serology or a commercially available genusspecific PCR assay. Culture has rarely been performed on human samples from patients with tick-borne rickettsioses so far. With regard to the literature available, data about Rickettsia species circulating in China are scarce, but of R. sibirica, R. heilongjiangensis with great importance for disease epidemiology. In the present study, we aimed to determine the prevalence of R. raoultii in D. silvarum, a predominant tick species throughout the pastroral and forest, which frequently bites outdoor tourists and residents in its seasons to reveal the potential threat for human of R. raoultii.
Material and method Tick sampled Dermacentor silvarum were collected from Heihe suburban area (50.160 N, 127.230 E), Heilongjiang province, China by swiping flags on vegetations. All specimens were identified by morphological characters with standard taxonomic keys. Genomic DNA extraction All ticks were processed individually for DNA extraction using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored at -20 °C a until analysis. Polymerase chain reaction Rickettsia DNA was detected by PCR with primers specific for rickettsiae as follows: Primer GltA.877p and GltA1258n (Alekseev and Dubinia 2003), which amplify a 382 bp part of gltA gene; Primer Rr17.61 and Rr17.492 (Noda et al. 1997), which amplify a 438 bp fragment of 17 kDa protein gene; Primer Rr70p and Rr602n (Regnery et al. 1991)
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for 530 bp fragment of ompA gene; RrompBf and RrompBr for a 515 bp fragment of ompB (Choi et al. 2005). The PCR were performed as described previously with distilled water instead of tick DNA template was used as a negative control. The newly obtained sequences were aligned with corresponding sequences retrieved from the GenBank database (http://www.ncbi.nlm.nih.gov/) using BioEdit v.7.0.5.3. The phylogenetic trees for the genes were constructed applying the neighbour-joining (NJ) algorithm implemented in the software package MEGA 5.20.
Result A total of 93 adult ticks collected were identified as D. silvarum with 47 males and 46 females. Samples positive for both gltA and ompA were considered SFG rickettsial species. Using this criterion, the DNA fragments of R. raoultii was detected in 30 of 94 D. silvarum (32.25 %), 13 in males (27.66 %), 17 in females (36.95 %) with no significant difference between sex; All of the positive samples were additionally investigated with primers targeting ompA gene, ompB gene, 17 kDa protein gene respectively and got positive results as expected. The amplicons were sequenced and compared with those deposited in GenBank. The nucleotide sequences of the PCR products of gltA gene for R. raoultii (KC566999) were identical to R. raoultii citrate synthase (gltA) gene (JX885455.1)(JQ792121.1)(JQ792119.1) with 99.95 % identities. The sequence coding a fragment of 17 kDa protein gene (KF564790) showed 99.0 % similarities to R. raoultii surface antigen gene (JX885457.1). And ompA gene (KF564791) exhibited 100 % similarities to R. raoultii outer membrane protein A (ompA) gene (JN400407.1). While OmpB gene (KF564792) exhibited 100 % similarities to R. raoultii strain Elanda-23/95 OmpB gene (EU036984.1) and R. raoultii strain Khabarovsk OmpB gene (DQ365798.1) in territories of the former Soviet Union. The new sequences were clustered into a separate R. raoultii branches of SFG (Fig. 1). And the homology levels of ompA, ompB, gltA and 17 kDa protein gene are within species thresholds for R. raoultii proposed by Fournier and Raoult (2009). Therefore, we consider the rickettsia agent as R. raoultii.
Discussion To date, genotypes of R. raoultii were detected at least in 13 different tick species belonging to six genus, including Dermacentor (Vitorino et al. 2007; Spitalska´ et al. 2012; Raoult et al. 2005; Stan´czak 2006; Nijhof et al. 2007; Dautel et al. 2006; Rumer et al. 2011; Tian et al. 2012), Ixodes (Jiang et al. 2005; Boldisˇ et al. 2008; Speck et al. 2012), Rhipicephalus (Merino et al. 2005), Haemaphysalis (Ma´rquez 2008), Amblyomma (Paddock et al. 2010) and Hyalomma (Shpynov et al. 2004). However, the presence of R. raoultii in one tick species does not prove its transmission capability. As a matter of fact, the low prevalence and less than one generation maintain capability in some other tick species, such as I. persulcatus, reflect the occasional infection reality and the minor importance of these tick species in the transmission of R. raoultii (Samoylenko et al. 2009). The reported rate of natural R. raoultii infection in Dermacentor species varies from 4 to 97 % depending on the species and habit structures. Rickettsia raoultii could be maintained in Dermacentor ticks for 4–7 generations with a high level of transovarial and transstadial transmission (Samoylenko et al. 2009). Dermacentor species should play a major role in transmission of R. raoultii to human. Principal hosts of mature Dermacentor ticks are
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livestock, whereas immature ticks feed on rodents, hares, and pika (Ochotona spp.). Adults have been described to overwinter on hosts which might be related to the reservoir of the rickettsia organism. D. silvarum might be another candidate vector of R. raoultii besides D. marginatus and D. reticulates for the later two species are not common seen species in these areas. Presence of R. raoultii in D. silvarum is of immense clinical relevance for its pathogenic to human. At first, the predominant D. silvarum has been recorded and reported frequently to bite residents or tourists in this area according the datas provided by local Center for
A
AF120028.1 R. sp. DnS14 strain DnS14 gltA HQ335153.1 R. aeschlimannii strain EgyRickHimp-El-Arish-13 gltA DQ365803.1 R. raoultii strain Marne gltA DQ365804.1 R. raoultii strain Khabarovsk gltA 44 HM149279.1 R. sp. PoTiR7dt gltA JQ792121.1 R. raoultii isolate LYG419 gltA JX885455.1 R. raoultii isolate MDJ1 gltA KC566999 R. raoultii gltA this study
27
JQ792119.1 R. raoultii isolate LYG244 gltA AF120029.1 R. sp. RpA4 strain RpA4 gltA 52
AY737684.1 R. marmionii strain KB gltA
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EF102236.1 R. parkeri strain At24 gltA 43 43 AF178035.1 R. sp. BJ-90 gltA DQ100162.1 R. peacockii strain Rustic gltA JQ727682.1 R. sp. T170-B gltA
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AB473812.1 R. heilongjiangensis strain: CH8-1 gltA DQ909073.1 R. japonica gltA U59715 R. prowazekii gltA 0.005
KF010848.1 R. raoultii isolate Dsm ompA
B
JX885458.1 R. raoultii isolate MDJ4 OmpA 46 AY093696.1 R. sp. JL-02 OmpA KF564791 R. raoultii ompA this study 64 JN400407.1 R. raoultii clone 7b ompA 32 JN400407.1 R. raoultii clone 7b ompA JN400406.1 R. raoultii clone 6b ompA HM161794.1 R. raoultii strain WB13/Dm R Casola Valsenio ompA 90 98
HM161792.1 R. raoultii strain WB14/Dm R Casola Valsenio ompA HM161789.1 R. raoultii strain WB16/Dm Monterenzio ompA
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AF120022.1 R. sp. RpA4 OmpA AF120018.1 R. sp. DnS28 OmpA EF194096.1 R. amblyommii strain Texas AM ompA EF689733.1 R. amblyommii isolate TX116 OmpA
100
CP003342.1 R. rhipicephali str. 3-7-female6-CWPP CP003319.1| R. massiliae str. AZT80
0.1
Fig. 1 Phylogenetic tree of Rickettsia raoultii based on (panel A): 381-bp the citrate synthase (gltA), (panel B) 533-bp the outer membrane protein A (ompA) gene, (panel C) 515-bp the outer membrane protein B (ompB) gene and (panel D) 438-bp 17 kDa protein gene. The tree was calculated by neighbor-joining method using MEGA 5.2 software. Values of the bootstrap support of the particular branching calculated for 10,000 replicates are indicated at the nodes. The variant sequences obtained in this study were designated by accession number and species and/or strain name. The sequence of R. prowazekii was used as an outgroup
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583 EU036984.1 R. raoultii strain Elanda-23/95 OmpB
C
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DQ365798.1 R. raoultii strain Khabarovsk OmpB KF564792 R. raoultii OmpB this study HQ232276.1 R. raoultii from Dermacentor reticulatus M161
97 HQ232278.1 R. raoultii from Dermacentor reticulatus Mue18 HQ232277.1 R. raoultii from Dermacentor reticulatus Mue9 HQ232275.1 R. raoultii from Dermacentor reticulatus M10
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HQ232274.1 R. raoultii from Dermacentor reticulatus F273 DQ365797.1 R. raoultii strain Marne OmpB 58
JQ792107.1 R. raoultii isolate XG86 92
61
53 74 75
AF123722.1 R. sibirica OmpB AY331393.1 R. sp. BJ-90
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CP002428.1 R. slovaca 13-B AF123706.1 R. africae OmpB AF123724 R. honei strain Thai OmpB CP003319.1R. massiliae str. AZT80 AF123716.1 R. montanensis OmpB JX683117.1 R. monacensis isolate I92 AF211821.1 R. prowazekii strain Virginia
0.05
D
CP003334.1 Candidatus R. amblyommii str. GAT-30V KF564790 R. raoultii 17 kD this study
13
EF392727.1 R. sp. RpA4 17 kDa 16
JX885457.1 R. raoultii isolate MDJ3 17 kD
22 57
AB114810.1 R. sp. Hj126 17-kDa AB114804.1 R. sp. Hf332 17-kDa
33 48
EF451002.1 R. sp. Argentina 17-kDa 83 CP003340.1 R. montanensis str. OSU 85-930 U11017.1 R. montana 17 kDa CP000683.1 R. massiliae MTU5
DQ865207.1 R. rhipicephali strain HJ5 17 kDa AY730679.1 R. prowazekii strain Breinl 17 kDa 0.01
Fig. 1 continued
Disease Control and Prevention. Secondly, R.raoultii was confirmed as one of etiological agents causing TIBOLA-DEBONEL symptoms (Parola et al. 2009). As previously stated, R.raoultii was found in a D. marginatus tick attacked patient in France in whom typical clinical symptoms of tick-borne lymphadenopathy developed (Mediannikov et al. 2008), and it was also detected by PCR in the blood of one patient in Spain with TIBLLA/ DEBONEL (Ibarra et al. 2006). The potential threat of R. raoultii should be considered in differential diagnosis in spotted fever patients. With regard to the literature available R. raoultii might encompasses a large geographical spread in China, including Jinlin (Cao et al. 2008), Tibet (Wang et al. 2012), Xinjiang and Gansu provinces (Tian et al. 2012), which indicate a significant number of human cases might occur. However, few rickettsioses cases of human caused by R. raoultii have been reported till now. Underdiagnosis of Rickettsioses cases in human might be
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attributed to lack of proper clinical diagnosis and effective surveillance system for tickborne diseases, people who infected with R. raoultii even don’t know what disease they have acquired. The other reason maybe R. raoultii was less pathogenic than other Rickettsia (Parola et al. 2009) though high prevalence in Dermacentor spp. Detail investigations about the pathogenic characters and the natural cycle of R. raoultii should be carried out in the future. Acknowledgments This study was supported by the National Science Foundation of China (30400364, 81271878), Special Fund of the Ministry of Health of P. R. China (Grant no. 201202019) for funding the research; Heilongjiang Import and Export Inspection and Quarantine Bureau autonomous scientific research subject (Grant no .2013HK005).
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Rickettsia raoultii, the predominant Rickettsia found in Dermacentor silvarum ticks in China–Russia border areas Jing Wen • Dan Jiao • Jian-hua Wang • De-hai Yao • Zhi-xiang Liu Gang Zhao • Wen-dong Ju • Cheng Cheng • Yi-jing Li • Yi Sun
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Received: 5 November 2013 / Accepted: 5 March 2014 / Published online: 4 April 2014 Ó Springer International Publishing Switzerland 2014
Abstract Since the year 2000, clinical patterns resembling tick-borne rickettsioses have been noticed in China–Russia border areas. Epidemiological data regarding species of the aetiological agent, tick vector prevalence and distribution as well as incidence of human cases in the areas are still sparse to date. In order to identify Rickettsia species occurring in the areas, we investigated Dermacentor silvarum collected in the selected areas. Rickettsia raoultii was the predominant Rickettsia found in D. silvarum evident with ompA, ompB, gltA and 17 kDa protein genes. The Rickettsia prevalence in D. silvarum appeared to be 32.25 % with no sex difference. The results extend the common knowledge about the geographic distribution of R. raoultii and its candidate vector tick species, which suggest an emerged potential threat of human health in the areas. Keywords
R. raoultii D. silvarum China–Russia border area
J. Wen Y. Li (&) College of Veterinary Medicine, Northeast Agricultural University, Harbin City, Heilongjiang Province, China e-mail: [email protected] J. Wen e-mail: [email protected]; [email protected] J. Wen D. Jiao J. Wang D. Yao Z. Liu G. Zhao Heihe Entry-Exit Inspection and Quarantine Bureau, Heihe City, Heilongjiang Province, China e-mail: [email protected] G. Zhao e-mail: [email protected] W. Ju C. Cheng Heilongjiang Entry-Exit Inspection and Quarantine Bureau, Harbin City, Heilongjiang Province, China Y. Sun (&) Department of Vector Biology and Control, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People’s Republic of China e-mail: [email protected]; [email protected]
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Introduction Rickettsia are obligate intracellular gram-negative bacteria which belong to the Spotted Fever Group (SFG) Rickettsiae. Rickettsia are associated with arthropod vectors and cause mild to fatal diseases in human (Fournier and Raoult 2009). In 1999, Rickettsia raoultii was found from Dermacentor nutallii in Siberia and Rhipicephalus pumilio in the Astrakhan region of former Soviet Union accoding to phylogenetic analysis of rrs (16s rDNA), Citrate synthase (gltA) and Outer membrane B (ompB) (Rydkina et al. 1999). In 2002, R. raoultii DNA was detected in a D. marginatus tick taken from the scalp of a patient in whom Tick-borne lymphadenopathy (TIBOLA) or Dermacentor-borne necrosis erythema lymphadenopathy (DEBONEL) developed in France (Mediannikov et al. 2008). And soon R. raoultii was recognized as one of caused agents of TIBOLA/DEBONEL (Parola et al. 2009), which is nowadays the most prevalent tick-borne rickettsiose in Europe Oteo and Portillo (2012). In China–Russia border areas, human cases of tick-borne rickettsioses have been increasing in recent years. From 2000 to 2012, over 500 cases were reported to National Center of Disease prevention and Control of China, most cases occur in northeast China, mainly in the China–Russia border areas. Most of the cases were diagnosed by clinical symptoms, and in some cases confirmed by serology or a commercially available genusspecific PCR assay. Culture has rarely been performed on human samples from patients with tick-borne rickettsioses so far. With regard to the literature available, data about Rickettsia species circulating in China are scarce, but of R. sibirica, R. heilongjiangensis with great importance for disease epidemiology. In the present study, we aimed to determine the prevalence of R. raoultii in D. silvarum, a predominant tick species throughout the pastroral and forest, which frequently bites outdoor tourists and residents in its seasons to reveal the potential threat for human of R. raoultii.
Material and method Tick sampled Dermacentor silvarum were collected from Heihe suburban area (50.160 N, 127.230 E), Heilongjiang province, China by swiping flags on vegetations. All specimens were identified by morphological characters with standard taxonomic keys. Genomic DNA extraction All ticks were processed individually for DNA extraction using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored at -20 °C a until analysis. Polymerase chain reaction Rickettsia DNA was detected by PCR with primers specific for rickettsiae as follows: Primer GltA.877p and GltA1258n (Alekseev and Dubinia 2003), which amplify a 382 bp part of gltA gene; Primer Rr17.61 and Rr17.492 (Noda et al. 1997), which amplify a 438 bp fragment of 17 kDa protein gene; Primer Rr70p and Rr602n (Regnery et al. 1991)
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for 530 bp fragment of ompA gene; RrompBf and RrompBr for a 515 bp fragment of ompB (Choi et al. 2005). The PCR were performed as described previously with distilled water instead of tick DNA template was used as a negative control. The newly obtained sequences were aligned with corresponding sequences retrieved from the GenBank database (http://www.ncbi.nlm.nih.gov/) using BioEdit v.7.0.5.3. The phylogenetic trees for the genes were constructed applying the neighbour-joining (NJ) algorithm implemented in the software package MEGA 5.20.
Result A total of 93 adult ticks collected were identified as D. silvarum with 47 males and 46 females. Samples positive for both gltA and ompA were considered SFG rickettsial species. Using this criterion, the DNA fragments of R. raoultii was detected in 30 of 94 D. silvarum (32.25 %), 13 in males (27.66 %), 17 in females (36.95 %) with no significant difference between sex; All of the positive samples were additionally investigated with primers targeting ompA gene, ompB gene, 17 kDa protein gene respectively and got positive results as expected. The amplicons were sequenced and compared with those deposited in GenBank. The nucleotide sequences of the PCR products of gltA gene for R. raoultii (KC566999) were identical to R. raoultii citrate synthase (gltA) gene (JX885455.1)(JQ792121.1)(JQ792119.1) with 99.95 % identities. The sequence coding a fragment of 17 kDa protein gene (KF564790) showed 99.0 % similarities to R. raoultii surface antigen gene (JX885457.1). And ompA gene (KF564791) exhibited 100 % similarities to R. raoultii outer membrane protein A (ompA) gene (JN400407.1). While OmpB gene (KF564792) exhibited 100 % similarities to R. raoultii strain Elanda-23/95 OmpB gene (EU036984.1) and R. raoultii strain Khabarovsk OmpB gene (DQ365798.1) in territories of the former Soviet Union. The new sequences were clustered into a separate R. raoultii branches of SFG (Fig. 1). And the homology levels of ompA, ompB, gltA and 17 kDa protein gene are within species thresholds for R. raoultii proposed by Fournier and Raoult (2009). Therefore, we consider the rickettsia agent as R. raoultii.
Discussion To date, genotypes of R. raoultii were detected at least in 13 different tick species belonging to six genus, including Dermacentor (Vitorino et al. 2007; Spitalska´ et al. 2012; Raoult et al. 2005; Stan´czak 2006; Nijhof et al. 2007; Dautel et al. 2006; Rumer et al. 2011; Tian et al. 2012), Ixodes (Jiang et al. 2005; Boldisˇ et al. 2008; Speck et al. 2012), Rhipicephalus (Merino et al. 2005), Haemaphysalis (Ma´rquez 2008), Amblyomma (Paddock et al. 2010) and Hyalomma (Shpynov et al. 2004). However, the presence of R. raoultii in one tick species does not prove its transmission capability. As a matter of fact, the low prevalence and less than one generation maintain capability in some other tick species, such as I. persulcatus, reflect the occasional infection reality and the minor importance of these tick species in the transmission of R. raoultii (Samoylenko et al. 2009). The reported rate of natural R. raoultii infection in Dermacentor species varies from 4 to 97 % depending on the species and habit structures. Rickettsia raoultii could be maintained in Dermacentor ticks for 4–7 generations with a high level of transovarial and transstadial transmission (Samoylenko et al. 2009). Dermacentor species should play a major role in transmission of R. raoultii to human. Principal hosts of mature Dermacentor ticks are
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livestock, whereas immature ticks feed on rodents, hares, and pika (Ochotona spp.). Adults have been described to overwinter on hosts which might be related to the reservoir of the rickettsia organism. D. silvarum might be another candidate vector of R. raoultii besides D. marginatus and D. reticulates for the later two species are not common seen species in these areas. Presence of R. raoultii in D. silvarum is of immense clinical relevance for its pathogenic to human. At first, the predominant D. silvarum has been recorded and reported frequently to bite residents or tourists in this area according the datas provided by local Center for
A
AF120028.1 R. sp. DnS14 strain DnS14 gltA HQ335153.1 R. aeschlimannii strain EgyRickHimp-El-Arish-13 gltA DQ365803.1 R. raoultii strain Marne gltA DQ365804.1 R. raoultii strain Khabarovsk gltA 44 HM149279.1 R. sp. PoTiR7dt gltA JQ792121.1 R. raoultii isolate LYG419 gltA JX885455.1 R. raoultii isolate MDJ1 gltA KC566999 R. raoultii gltA this study
27
JQ792119.1 R. raoultii isolate LYG244 gltA AF120029.1 R. sp. RpA4 strain RpA4 gltA 52
AY737684.1 R. marmionii strain KB gltA
43
EF102236.1 R. parkeri strain At24 gltA 43 43 AF178035.1 R. sp. BJ-90 gltA DQ100162.1 R. peacockii strain Rustic gltA JQ727682.1 R. sp. T170-B gltA
60
AB473812.1 R. heilongjiangensis strain: CH8-1 gltA DQ909073.1 R. japonica gltA U59715 R. prowazekii gltA 0.005
KF010848.1 R. raoultii isolate Dsm ompA
B
JX885458.1 R. raoultii isolate MDJ4 OmpA 46 AY093696.1 R. sp. JL-02 OmpA KF564791 R. raoultii ompA this study 64 JN400407.1 R. raoultii clone 7b ompA 32 JN400407.1 R. raoultii clone 7b ompA JN400406.1 R. raoultii clone 6b ompA HM161794.1 R. raoultii strain WB13/Dm R Casola Valsenio ompA 90 98
HM161792.1 R. raoultii strain WB14/Dm R Casola Valsenio ompA HM161789.1 R. raoultii strain WB16/Dm Monterenzio ompA
44
AF120022.1 R. sp. RpA4 OmpA AF120018.1 R. sp. DnS28 OmpA EF194096.1 R. amblyommii strain Texas AM ompA EF689733.1 R. amblyommii isolate TX116 OmpA
100
CP003342.1 R. rhipicephali str. 3-7-female6-CWPP CP003319.1| R. massiliae str. AZT80
0.1
Fig. 1 Phylogenetic tree of Rickettsia raoultii based on (panel A): 381-bp the citrate synthase (gltA), (panel B) 533-bp the outer membrane protein A (ompA) gene, (panel C) 515-bp the outer membrane protein B (ompB) gene and (panel D) 438-bp 17 kDa protein gene. The tree was calculated by neighbor-joining method using MEGA 5.2 software. Values of the bootstrap support of the particular branching calculated for 10,000 replicates are indicated at the nodes. The variant sequences obtained in this study were designated by accession number and species and/or strain name. The sequence of R. prowazekii was used as an outgroup
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583 EU036984.1 R. raoultii strain Elanda-23/95 OmpB
C
88
DQ365798.1 R. raoultii strain Khabarovsk OmpB KF564792 R. raoultii OmpB this study HQ232276.1 R. raoultii from Dermacentor reticulatus M161
97 HQ232278.1 R. raoultii from Dermacentor reticulatus Mue18 HQ232277.1 R. raoultii from Dermacentor reticulatus Mue9 HQ232275.1 R. raoultii from Dermacentor reticulatus M10
76
HQ232274.1 R. raoultii from Dermacentor reticulatus F273 DQ365797.1 R. raoultii strain Marne OmpB 58
JQ792107.1 R. raoultii isolate XG86 92
61
53 74 75
AF123722.1 R. sibirica OmpB AY331393.1 R. sp. BJ-90
83
CP002428.1 R. slovaca 13-B AF123706.1 R. africae OmpB AF123724 R. honei strain Thai OmpB CP003319.1R. massiliae str. AZT80 AF123716.1 R. montanensis OmpB JX683117.1 R. monacensis isolate I92 AF211821.1 R. prowazekii strain Virginia
0.05
D
CP003334.1 Candidatus R. amblyommii str. GAT-30V KF564790 R. raoultii 17 kD this study
13
EF392727.1 R. sp. RpA4 17 kDa 16
JX885457.1 R. raoultii isolate MDJ3 17 kD
22 57
AB114810.1 R. sp. Hj126 17-kDa AB114804.1 R. sp. Hf332 17-kDa
33 48
EF451002.1 R. sp. Argentina 17-kDa 83 CP003340.1 R. montanensis str. OSU 85-930 U11017.1 R. montana 17 kDa CP000683.1 R. massiliae MTU5
DQ865207.1 R. rhipicephali strain HJ5 17 kDa AY730679.1 R. prowazekii strain Breinl 17 kDa 0.01
Fig. 1 continued
Disease Control and Prevention. Secondly, R.raoultii was confirmed as one of etiological agents causing TIBOLA-DEBONEL symptoms (Parola et al. 2009). As previously stated, R.raoultii was found in a D. marginatus tick attacked patient in France in whom typical clinical symptoms of tick-borne lymphadenopathy developed (Mediannikov et al. 2008), and it was also detected by PCR in the blood of one patient in Spain with TIBLLA/ DEBONEL (Ibarra et al. 2006). The potential threat of R. raoultii should be considered in differential diagnosis in spotted fever patients. With regard to the literature available R. raoultii might encompasses a large geographical spread in China, including Jinlin (Cao et al. 2008), Tibet (Wang et al. 2012), Xinjiang and Gansu provinces (Tian et al. 2012), which indicate a significant number of human cases might occur. However, few rickettsioses cases of human caused by R. raoultii have been reported till now. Underdiagnosis of Rickettsioses cases in human might be
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attributed to lack of proper clinical diagnosis and effective surveillance system for tickborne diseases, people who infected with R. raoultii even don’t know what disease they have acquired. The other reason maybe R. raoultii was less pathogenic than other Rickettsia (Parola et al. 2009) though high prevalence in Dermacentor spp. Detail investigations about the pathogenic characters and the natural cycle of R. raoultii should be carried out in the future. Acknowledgments This study was supported by the National Science Foundation of China (30400364, 81271878), Special Fund of the Ministry of Health of P. R. China (Grant no. 201202019) for funding the research; Heilongjiang Import and Export Inspection and Quarantine Bureau autonomous scientific research subject (Grant no .2013HK005).
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