Eur J Clin Microbiol Infect Dis (2012) 31:365–369 DOI 10.1007/s10096-011-1318-7
ARTICLE
Diplorickettsia massiliensis as a human pathogen G. Subramanian & O. Mediannikov & E. Angelakis & C. Socolovschi & G. Kaplanski & L. Martzolff & D. Raoult
Received: 28 April 2011 / Accepted: 7 June 2011 / Published online: 22 July 2011 # Springer-Verlag 2011
Abstract Diplorickettsia massiliensis has been recently isolated from Ixodes ricinus ticks. We screened 13,872 serum samples from patients in France with suspected tick-borne diseases and found three cases that had serological evidence of D. massiliensis infection; for one of these three cases, we obtained molecular evidence of an infection as well.
Ehrlichia spp., and Francisella tularensis, as well as a number of viruses (such as tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, etc.) and protozoa (Babesia microti) [2]. R. monacensis and R. helvetica were first isolated from ticks and considered to be nonpathogenic. Later, evidence that both are pathogenic for humans were published [3]. In this study, we report the first evidence of D. massiliensis infection.
Introduction Materials and methods Ixodid ticks are obligate hematophagous arthropods that parasitize vertebrates. Diplorickettsia massiliensis was originally isolated from Ixodes ricinus ticks collected in Slovakia. They are gram-negative, non-spore-forming small rods that belong to the γ-proteobacteria class and are related to Rickettsiella spp. [1]. Almost all bacteria isolated from I. ricinus are pathogenic for humans, notably, Borrelia spp. (B. burgdorferi, B. afzelii, and B. garinii), Rickettsia helvetica, R. monacensis, Anaplasma phagocytophilum, G. Subramanian : O. Mediannikov : E. Angelakis : C. Socolovschi : D. Raoult (*) Unité des Rickettsies, Faculté de Médecine, Université de la Méditerranée, URMITE, UMR CNRS 6236—IRD198, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France e-mail:
[email protected] G. Kaplanski Service de Médicine Interne, CHU Conception, Marseille, France L. Martzolff Service de Médecine Interne et Enodocrinologie, Hôpital Emile Muller, 20 Avenue du Dr René Laennec, BP 1370, Mulhouse Cedex, France
D. massiliensis was cultured in XTC-2 cells with Leibovitz (Invitrogen) supplemented with 2% fetal bovine serum; a 2% tryptose phosphate broth solution was used for culture (Sigma-Aldrich, Ayrshire, UK) [1]. Purified bacteria were washed in PBS (10,000g, 10 min) and stored at −80°C until use. Each step of the purification was monitored by Gimenez staining. Polyclonal antibodies were produced in six- to eight-week-old female BALB/c mice that were intraperitoneally inoculated with 106 D. massiliensis bacteria in CpG adjuvant (three boosts in total). Mice were sacrificed on day 40 post-infection and the whole blood was used to obtain polyclonal antibodies. We tested serum samples from patients with suspected tick-borne diseases routinely received in our laboratory from July 2009 to June 2010. Sera were obtained from both hospitalized patients and outpatients in France. Not all of them had the history of tick bite. However, in many cases of proven tick bite infection, no tick bites are often noted by the patient. In total, 13,872 serum samples were screened by immunofluorescence assays (IFAs) in three dilutions (1/25, 1/50, and 1/100) for the presence of IgG and IgM against D. massiliensis. IgG titers of ≥1:100 and IgM titers of ≥1:50 were considered to be positive. All serum samples were
366 Fig. 1 Western blot (WB) of Diplorickettsia massiliensis with mouse polyclonal antiserum compared with WB of three patients’ sera: a D. massiliensis; b patient 1; c patient 2; d patient 3. Serum dilution used for the Diplorickettsia strain of mice: 1:5,000 dilution; serum dilution used for patients: 1:1,000 dilution
Eur J Clin Microbiol Infect Dis (2012) 31:365–369 kDa
a
kDa
c
d
105.2 105.2
84.2
84.2 50.4 50.4 36.8 36.8 29.0 20.5
also tested by IFAs for the presence of antibodies against Bartonella spp., F. tularensis, Rickettsia spp., Coxiella burnetii, and D. massiliensis. The studies were approved by the local ethical committee (Marseille, France). We identified three patients (0.02%) with sera positive for D. massiliensis by IFAs, and the results were confirmed by Western blot (WB). WB was firstly performed using the mouse anti-D. massiliensis sera, as described elsewhere [4]. We found that the 32-kDa protein was immunogenic. Several reactive unknown proteins around 20 kDa (Fig. 1a) were also identified. The sera of the three patients Fig. 2 Two-dimensional Western blot (2-DWB) of patient 2 (6–11, 13 cm). The arrows indicate the D. massiliensis characteristic proteins (data not shown)
b
kDa
105.2
84.2
50.4
36.8
29.0
20.5
32 kDa 20 kDa
29.0
20.5
20 kDa
were first analyzed by WB, and we identified bands corresponding to the unknown 20-kDa proteins of D. massiliensis (Fig. 1, lanes b–d). Subsequently, the sera were analyzed by two-dimensional WB (2-DWB). Only the 2-DWB picture of the second patient sample was closely related to Diplorickettsia proteins (Fig. 2). Patient 1 was also positive for F. tularensis and B. henselae, and WB confirmed the co-infection with these agents. Even though other WB results highly resembled the result with D. massiliensis infection, these patients’ sera only showed scattered, low-intensity spots in 2-DWB. DNA was
Eur J Clin Microbiol Infect Dis (2012) 31:365–369 Table 1 Primers and probes
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Primer name
Gene
Sequence
RpoBf
rpoB
CCG-CGC-AAA-AAG-TTG-ATC-T
RpoBR
rpoB
GTA-AGT-CCG-CAA-GAC-GAA-GC
Topof Topor
Topoisomerase IV subunit A Topoisomerase IV subunit A
TAT-TAC-CGG-CAC-GTC-TAC-C GAG-GCC-ACT-TCA-CGA-AGA-T-
Probe RpoBP
rpoB
6-FAM- CCA-GAC-AAT-GAG-TTG-CTC-GA-TAMRA
TopoP
Topoisomerase IV subunit A
6 FAM- ATA-TCT-TAC-TCA-ACG-GCG-CC-TAMRA
extracted from the positive sera using a QIAamp Tissue Kit (QIAGEN, Valencia, CA, USA) and was used as a template for real-time polymerase chain reaction (RT-PCR) that targets specific portions of the D. massiliensis rpoB gene and the topoisomerase IV subunit A (Table 1). RT-PCR for patient 2 was positive for the rpoB gene but negative for the topoisomerase IV subunit A. RT-PCR was negative for the other two patients. Detailed histories of the three patients are described below (Table 2).
Case report Patient 1 was a 54-year-old woman from Marseille, France, with a skin eschar on her elbow that appeared on September 2009. She did not show lymphadenopathy or other clinical signs. She had a pet dog and did not recall any tick bites. Her blood cell counts, biochemical analysis, and C-reactive protein (CRP) were normal. The eschar disappeared after 10 days without treatment.
Table 2 Patient data
Fever Skin eschar Lymphadenopathy Rash Other symptoms Insect bite Animal contact Travel Other Blood analysis White blood cells Hemoglobin Platelets ALAT ΑSΑΤ γGT CRP IFA (titer)
Western blot
2-D Western blot RT-PCR for rpoB RT-PCR for topoisomerase IVsubunit A
Patient 1
Patient 2
Patient 3
No Yes No No No No Dog No No 6,500 13.4 g/dl 287,000 Normal Normal Normal
39°C No No No Arthralgia, myalgia, and dyspnea No No Slovenia No 20,000 (50% neutrophils) 6 g/dl 74,000 101 77 123 U/l
No No No No Arthralgia and myalgia Yes Cat No Hepatic steatosis 9,330 13.7 g/dl 290,000 130 160 60
Normal D. massiliensis(IgG 1:400) F. tularensis (IgG1:400) B. henselae (IgG1:400) R. felis (IgG1:512) D. massiliensis F. tularensis B. henselae Weak reactive Negative Negative
67 mg/l D. massiliensis(IgG 1:100)
Normal D. massiliensis(IgG 1:400)
D. massiliensis
D. massiliensis
Proteins characteristic for Diplorickettsia Positive (33 ct) Negative
Weak reactive Negative Negative
368
Patient 2 was a 71-year-old male from Marseille, France, who presented with long-lasting fever (39°C), asthenia, and dyspnea in October 2009. One week before the symptoms arose, the patient traveled and stayed for a week in a forest in Slovenia. He did not mention animal contact or tick bites. For 10 years, this patient had suffered from cardiac insufficiency and had a renal artery stent. His history included multiple episodes of relapsing fever. During examination, the patient was tachycardic (180 beats/min), with no signs of pulmonary infection or lymphadenopathy. Blood analysis revealed increased white blood cells, anemia, thrombocytopenia, increased levels of transaminases, and increased CRP. Patient 3 was a 29-year-old female from Marseille, France, who was a cat owner and presented with arthralgia and myalgia in August 2009. She mentioned that, before the symptoms started, she was bitten by an insect on her elbow. Clinical examination showed serious arthralgias and cramps in the wrists. Blood analyses showed increased levels of transaminases, whereas her blood cell counts and CRP were normal. Ultrasound echography revealed a hepatic steatosis, whereas liver biopsy was normal. Anti-nuclear, mitochondrial, smooth muscle, liver, and kidney antibodies were negative, thus, excluding autoimmune diseases.
Discussion In the present study, we screened serum samples from patients with suspected tick-borne diseases by IFAs and detected species-specific antibodies against D. massiliensis in three cases (0.02% of all of the tested sera). We identified an immunogenic protein around 32 kDa in size, characteristic of D. massiliensis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and two protein spots around 20 and 28 kDa in size on a 2-D map of D. massiliensis. However, we were unable to characterize these proteins because the D. massiliensis genome sequence is not yet complete. By WB and 2-DWB, we confirmed the presence of antibodies against D. massiliensis proteins. Moreover, PCR for D. massiliensis showed a positive result for one patient. All three patients had indirect epidemiological and clinical evidence of tick contact, including an eschar-like skin lesion, a history of arthropod bites and an experience of staying in a forest. The clinical features of the three reported cases do not characterize a clear clinical entity; however, patient 1 presented a skin eschar, which is typical of several tickborne diseases. In the last few years, several new tick-borne diseases have been identified, including the diseases caused by B. burgdorferi sensu lato, B. lonestari, E. chaffeensis, E. ewingii, and A. phagocytophilum [5–8]. Moreover, in a
Eur J Clin Microbiol Infect Dis (2012) 31:365–369
European study of acute febrile illnesses after tick bites, only 39% of 130 patients had a proven etiology of their illnesses. Similar data have been reported in the USA, where 27 to 45% patients had a proven etiology [9–10]. Our strategy to screen samples, such as skin biopsies and sera, from patients with suspected tick bites or ticks is critical in detecting new infectious pathogens and has already helped us detect human pathogens that were originally considered as non-pathogenic, such as R. raoultii [12], R. helvetica [13], and R. sibirica mongolitimonae [14]. However, more clinical and serological investigations are required in order to further confirm D. massiliensis as a human pathogen. Acknowledgments The authors thank Malgorzata Kowalczewska for the helpful technical assistance. Geetha Subramanian is a receiver of a stipend from APHM (Assistance Publique—Hôpitaux de Marseille). No funding of any kind has been received and all data have been generated as part of the routine work at URMITE, UMR CNRS 6236—IRD198. We declare that we have no competing interests. Conflict of interest None.
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