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Bartonella quintana is a fastidious gram-negative bacterium that is regarded as a reemerging human pathogen (1) and is responsible for various human diseases (12). Although trench fever, the first clinical manifestation of B. quintana infection to be recognized (13), affected thousands of soldiers during World Wars I and II, medical interest in trench fever waned for almost 30 years because the disease was only rarely encountered. In the 1990s, B. quintana was identified as an agent of bacillary angiomatosis in AIDS patients (17), endocarditis (5, 16, 18), chronic bacteremia (3, 19), and chronic lymphadenopathy (15). These diseases are associated with homelessness or cramped, unhygienic circumstances, together with cold weather and the presence of body lice. The role of lice had been observed as early as 1920 (4). Various experiments have demonstrated that the disease could be induced in human volunteers (20) and Macacus rhesus monkeys (14) by injection of B. quintana and that the bacterium multiplied in the gut lumen of naturally (21) or intrarectally (8) infected lice without interfering with viability and was excreted in their feces (4, 8). However, despite growing interest in louse-transmitted diseases, there is no currently available experimental model to describe the relationship between B. quintana and the louse.

GFP-expressing B. quintana. B. quintana strain Oklahoma (ATCC 49793) was obtained from the American Type Culture Collection (Rockville, Md.) and cultivated as previously described (10). Plasmid pJMBGFP, containing the B. bacilliformis flagellin promoter and a Rep origin of replication obtained from pBBR1-MCS2 (9) (Table 1), was provided by James M. Battisti (Division of Biological Sciences, University Montana, Missoula, Mont.). It was extracted and purified from Escherichia coli using a Midi Prep kit (Qiagen, Inc., Chatsworth, Calif.) and then adjusted to 0.5 µg/ml. Transformation procedures were as previously described (6). The transformed bacteria were cultivated on 5% sheep blood agar containing 25 µg of kanamycin sulfate per ml and were further verified as B. quintana by PCR amplification of the 16S-23S rDNA intergenic spacer (its) followed by sequencing (6). The presence of plasmid DNA was checked as previously described (6).

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Experimental body louse infection. Body lice (Pediculus humanus corporis, strain Orlando) were kindly provided by D. Richard-Lenoble (Laboratoire de Parasitologie, Faculté de Médecine, Tours, France). A colony of lice was established and nourished daily on the shaved abdomen of specific-pathogen-free (SPF) New Zealand White rabbits (Fig. 1a). The lice were shown to be free from B. quintana by periodic its PCR amplification of samples from their gut and feces. One SPF rabbit, designated R1, was first injected intravenously with 2.5 ml of a solution of 10 mg of kanamycin (Sigma, St. Louis, Mo.) per ml, and 15 min later was injected with 20 ml of a suspension of 10⁶ CFU of pJMBGFP-expressing B. quintana per ml in saline, before 800 15-day-old lice, half of which were female, were allowed to feed on its abdomen. The lice were then kept at 30°C and 70% humidity. The day of infection was referred to as day 1. On the following days, lice were allowed to feed daily on a second SPF rabbit designated R2. Before the lice were allowed to feed each day, R2 was injected intravenously each day with 2.5 ml of 10 mg of kanamycin sulfate (Sigma) per ml. To detect B. quintana, its amplification by PCR and culture on kanamycin-containing agar for up to 60 days at 37°C under a 5% CO₂ atmosphere were performed on blood drawn from the infected rabbit after the lice had fed then every day for 5 days, and then once a week for 2 months. Similar tests were performed on 15 lice harvested after feeding on the infected rabbit, 15 lice harvested daily for 1 week and twice a week after that until no lice from the first generation remained, and, when present, eggs, larvae, and feces. Blood samples drawn from the infected rabbit (R1) were positive for B. quintana by culture and PCR on days 1 and 2, with 2 × 10⁴/ml and 7 × 10² CFU/ml, respectively, but were negative on day 3 and at all later times. On follow-up, the rabbit did not present with any abnormal symptoms but developed an anti-B. quintana immunoglobulin G response by day 15, with a titer of 1:400 as determined by indirect immunofluorescence. Blood drawn from rabbit R2 was negative by both culture and PCR, and they did not seroconvert. GFP-expressing B. quintana was grown and PCR amplified from all groups of lice. Between 7 and 20 CFU of Bartonella (mean ± standard deviation = 16.3 ± 2.9) was obtained from each louse (Fig. 1b). The number of CFU did not decrease over time. B. quintana was also grown and PCR amplified from fecal samples harvested throughout the experiment. Qualitative but not quantitative analysis of the colonies grown from infected feces was performed. Only fluorescent colonies grew. The GFP marker was therefore very useful for directly identifying B. quintana colonies (Fig. 1B). None of the uninfected control lice were positive by culture or PCR analysis. The average daily number of eggs from the infected group was 682.8 ± 335.1 and was not significantly different from the 705.2 ± 357.9 observed in the control group (P = 0.51). No growth or PCR amplification of B. quintana was obtained from the 300 eggs and 100 larvae tested. Unfortunately, we observed that body lice were autofluorescent, and therefore detection of GFP-expressing Bartonella was not possible (Fig. 1c).

View larger version (96K): [in this window] [in a new window]   FIG. 1.   Experimental model of body louse infection by GFP-expressing B. quintana. (a) Pediculus humanus humanus feeding on a rabbit; (b) fluorescent colony isolated on selective agar from an infected louse 10 days after the initial infection; (c) autofluorescence of body lice; (d) immunohistological detection of B. quintana. Note the numerous erythrocytes and the clusters of bartonellae in the intestine lumen (red clumps) and against the intestine wall (blue). Streptavidin-biotin-peroxidase method, polyclonal rabbit anti-B. quintana used at a dilution of 1:400, hemalun counterstain. Magnification, ×660.

View larger version (9K): [in this window] [in a new window]   FIG. 2.   Comparison of the survival rate over time of 200 lice infected with B. quintana and 200 control lice. Day 0, day of infection.