In testing paired serum samples from 40 consecutive cases of African tick bite fever, we detected diagnostic antibodies against spotted fever group rickettsiae in 45% of the patients by immunofluorescence assay (IFA) and in 100% of the patients by Western blotting (WB) (P < 0.01). A specific diagnosis of Rickettsia africae infection could be established in 15% of the patients by IFA and in 73% of the patients by a combination of WB and cross-adsorption assays (P < 0.01).African tick bite fever (ATBF) is a flu-like illness frequently accompanied by inoculation eschars, headache, and neck myalgia (7). The disease is caused by Rickettsia africae, a recently identified spotted fever group (SFG) rickettsia, and is transmitted by cattle ticks in large parts of rural sub-Saharan Africa (9). ATBF typically occurs in clusters and has recently emerged as a common cause of acute febrile illness in international visitors to the region (2,4,8,13,14). Serology is the most widely used microbiological method for diagnosing rickettsial infections. Several tests are available, but with the exception of immunofluorescence assay (IFA), Western blotting (WB), and cross-adsorption assay, most are not recommended (10, 11). IFA, which can detect immunoglobulin G (IgG) and IgM antibodies, has a sensitivity rate ranging from 84 to 100% (1,6,13,15). Poor species specificity is a major drawback, but this may be circumvented by using a multiple-antigen IFA where reactions to several species can be compared directly (4, 13). WB detects both early occurring antibodies against nonspecific lipopolysaccharide (LPS) antigens and late occurring antibodies against specific protein antigens (SPAs) located in the rickettsial outer membrane (15). In cases where WB is unable to identify the causative species, a cross-adsorption assay followed by a second WB on the resulting supernatant may be conclusive (11).(Part of this paper was presented at the 14th European Congress of Clinical Microbiology and Infectious Diseases, Prague, Czech Republic, 1 to 4 May 2004.) Eighty serum samples collected between days 1 and 45 after the onset of symptoms from 40 consecutive patients with ATBF were obtained from a prospective cohort study of tropical fevers in Norwegian safari travelers to rural sub-Saharan Africa in 1999 to 2001 (8). All cases met the epidemiological and clinical criteria for ATBF, including myalgia and fever commencing no later than 10 days after a safari, and 26 (65%) cases occurred in clusters. Twenty-nine (73%) patients were males, and the mean age was 38.3 years (range, 14 to 57 years). Nineteen (48%) patients were treated with doxycycline, one (3%) had a complicated course (reactive arthritis), and one (3%) was hospitalized.A multiple-antigen IFA was performed as previously reported (4), using seven SFG rickettsial antigens: R. africae strain ESF-5, R. conorii strain seven (Malish) ATCC VR-613T, Rickettsia sibirica mongolotimonae strain HA-91T, Rickettsia aeschlimannii strain MC16T, Rickettsia massiliae strain Mtu1T, Rickettsia akari strain MK...