Patient, materials and methods
On May 8, 2004, a 51-year-old man living in Barcellona Pozzo di Gotto, Messina, Sicily, Italy was admitted to the hospital with a 3-month fever (37.2°C), arthralgia, myalgia, malaise, pallor, stiffnes of hands and knee, nausea, low appetite and weight loss. The patient did not recall a tick bite but he visited hunting areas 15 days before the fever started and kept exotic birds (golden pheasants, partridges and guinea fowls) at his house for two years. The patient showed abnormal liver (462 mU/ml alkaline phosphatase, normal range (nr) = 98–280; 1.20 mg/dl total bilirubin, nr = 0.16–1.10; 0.35 mg/dl direct bilirubin, nr = 0.0–0.2) and renal (1.5 mg/dl creatinine, nr = 0.5–1.2) functions and low hemoglobin (9.9 g/dl; nr = 12–18). Pulmonary, cardiac and immunological involvement was absent. Cell counts and serum C-reactive protein levels were normal.
On September 13, 2004 the patient was admitted to the hospital for a second time. In addition to previous findings, the patient showed a mild splenomegaly, hemorrhoids, osteopenia, hiatal hernia, gastritis and inflammation of duodenum. Systemic inflammatory pathology and neoplasia were ruled out. Doctors suspected a depressive syndrome and the patient was treated with antidepressants.
Serum antibodies were determined for Lyme borreliosis, Rickettsia conorii, Coxiella burnetii and A. phagocytophilum (Fuller Laboratories, Fullerton, CA, USA), and Ehrlichia chaffeensis and A. phagocytophilum (Focus Technologies, Cypress, CA, USA) following manufacturer's recommendations. FITC-conjugated anti-human IgG (Sigma, St. Louis, MO, USA) and IgM (Aliphadia S.A., Wavre, Belgium) were used as secondary antibodies. The immunofluorescence tests for A. phagocytophilum use antigens derived from human HL-60 cells infected with the HGE-1 isolate and samples were considered negative when no fluoresce was detected at 1:80 (Fuller Laboratories) or 1:64 (Focus Technologies) dilution of the test serum, as recommended by the manufacturer. DNA was purified using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma) or TriReagent (Sigma) from patient's blood samples collected in September 8 and October 8, 2004. PCR analysis for R. conorii, C. burnetii, E. chaffeensis, Theileria spp., Babesia spp. and Anaplasma spp. were conducted on both DNA samples as previously described [8–11]. PCRs were conducted with 1 μl (0.1–10 ng) DNA using 10 pmol of each primer in a 50-μl volume (1.5 mM MgSO4, 0.2 mM dNTP, 1 × AMV/Tfl 5 × reaction buffer, 5u Tfl DNA polymerase) employing the Access RT-PCR system (Promega, Madison, WI, USA). Reactions were performed in an automated DNA thermal cycler for 35 cycles. PCR products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 Kb DNA Ladder, Promega). Control reactions were done without the addition of DNA to rule out contaminations during PCR.
Amplified Anaplasma 16S rDNA fragments were resin purified (Wizard, Promega) and cloned into pGEM-T vector (Promega) for sequencing both strands by double-stranded dye-termination cycle sequencing (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University). Two independent clones were sequenced from each PCR. Multiple sequence alignment was performed with the program AlignX (Vector NTI Suite, version 5.5; InforMax, North Bethesda, MD, USA).
