Isolation of Bartonella (Rochalimaea) henselae: effects of methods of blood collection and handling.

Bartonella (Rochalimaea) henselae causes cat-scratch disease, bacillary angiomatosis, peliosis hepatis, and fever in humans. B. henselae can be difficult to culture axenically, and as many as 5 weeks may be required before colonies are visible. We compared how different methods of blood collection and handling affect isolation of this pathogen. Blood specimens from B. henselae-infected cats were collected in both EDTA and Isolator blood-lysis tubes and were subsequently plated onto rabbit blood-brain heart infusion agar by using three different schedules: plating immediately, plating after 24 h at 25 degrees C, and plating after 26 days at -65 degrees C. Colonies were counted 14 and 35 days after plating. Blood collected in tubes containing EDTA, frozen at -65 degrees C, and then plated on blood agar yielded a median of 60,000 CFU/ml, compared with 25,333 CFU/ml after collection in the Isolator tubes (P < 0.01). Frozen blood yielded the largest number of B. henselae colonies for any of the schedules tested. These results support previous observations that the Isolator system is more sensitive than tubes containing EDTA for isolation of B. henselae and suggest that, for cat blood, collection in tubes containing EDTA and subsequent freezing may further improve the sensitivity of detection of B. henselae.

In vitro susceptibilities of Bartonella henselae, B. quintana, B. elizabethae, Rickettsia rickettsii, R. conorii, R. akari, and R. prowazekii to macrolide antibiotics as determined by immunofluorescent-antibody analysis of infected Vero cell monolayers.

The in vitro susceptibilities of Bartonella (Rochalimaea) henselae, B. quintana, B. elizabethae, Rickettsia akari, R. conorii, R. prowazekii, and R. rickettsii to different concentrations of azithromycin, clarithromycin, dirithromycin, erythromycin, and roxithromycin in Vero cell cultures were evaluated. Bartonella and Rickettsia spp. were allowed to initiate infection of the antibiotic-free Vero cell monolayers, which were maintained in 16-chamber microscope slides in the absence of antibiotics at 32 degrees C in a CO2-enriched atmosphere. The monolayers were then incubated for 3 h to allow for initial host cell intracellular penetration by infecting species. After inoculation, inocula were replaced and tested with media containing 12 different concentrations of each antibiotic in replicate (10 wells of each antibiotic dilution) for each species, and the monolayers were reincubated. Tetracycline served as the control. Growth status of Bartonella spp. and Rickettsia spp. was determined by evaluation of immunofluorescent staining bacilli. Five days later, when antibiotic-free, control-infected cell monolayers demonstrated significant fluorescence, media were removed for all cell monolayers, the monolayers were fixed, and all specimens were stained with standard indirect immunofluorescent antibody reagents. Fluorescent foci were enumerated by counting such foci on random fields visualized with an epifluorescence microscope. The extent of antibiotic-induced focus inhibition was recorded for each dilution of antibiotic and compared with that of an antibiotic-negative control. Effective antibiotic dilution endpoints for inhibition of Bartonella and Rickettsia proliferation, as judged by absence of increase of significant fluorescence (as compared with no-growth controls), were enumerated by determining the number of cell culture chambers at various antibiotic dilutions that were negative or positive for significant Bartonella- or Rickettsia-specific fluorescence. All of the macrolide agents tested were readily active against all three Bartonella organisms, and azithromycin, clarithromycin, and roxithromycin may have potential in the treatment of Rickettsia infections. Animal model-based clinical trials are warranted to define the specific treatment role of the newer macrolide antibiotics.

Experimentally induced Bartonella henselae infections followed by challenge exposure and antimicrobial therapy in cats.

OBJECTIVES: To elucidate kinetics of Bartonella henselae bacteremia and IgG response, evaluate antibiotic therapy, and investigate challenge exposure in cats. ANIMALS: Specific-pathogen-free cats. PROCEDURE: Cats were inoculated with B henselae or B quintana and monitored. Convalescent cats were challenge exposed with B henselae. Amoxicillin, enrofloxacin, erythromycin, and tetracycline HCl were evaluated for effect on B henselae bacteremia. RESULTS: Cats developed B henselae bacteremia within 1 week; bacteremia persisted for longer than 2 months before subsiding spontaneously. IgG antibody titer developed shortly after onset of bacteremia; antibody co-existed with bacteremia for several weeks and remained detectable after bacteremia subsided. Cats inoculated with B quintana remained abacteremic. On challenge exposure to B henselae, cats previously infected with B henselae remained abacteremic; cats previously inoculated with B quintana supported B henselae infection. Tetracycline HCl and erythromycin depressed B henselae bacteremia; however, duration of bacteremia remained similar to that in untreated cats. Obvious signs of illness were not observed. CONCLUSIONS: Long-duration, high-titer B henselae infections were highly reproducible in cats. Convalescent cats were immune to reinfection. B quintana-inoculated cats did not have evidence of infection and were susceptible to B henselae challenge exposure. Antibiotic therapy was incompletely efficacious in terminating cat bacteremia. CLINICAL RELEVANCE: A cat with an inapparent B henselae infection must provisionally be regarded as a possible reservoir for infection for a minimum of 2 to 3 months. Convalescent cats are resistant to reinfection. Usual antibiotic therapy was not completely efficacious. Measurement of IgG antibody can be used to detect past or current infection.