ANTIMICROBIAL AGENTS
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CHEMOTHERAPY, Nov. 1991,
p.
Vol. 35, No. 11
2375-2381
0066-4804/91/112375-07$02.00/0 Copyright © 1991, American Society for Microbiology
Efficacy of Chloramphenicol, Enrofloxacin, and Tetracycline for Treatment of Experimental Rocky Mountain Spotted Fever in Dogs EDWARD B. BREITSCHWERDT,1* MICHAEL G. DAVIDSON,' DAVID P. AUCOIN,2 MICHAEL G. LEVY,3 NANCY S. SZABADOS,1 BARBARA C. HEGARTY,1 ANITA L. KUEHNE,l AND ROBERT L. JAMES4 Departments of Companion Animal and Special Species Medicine,' Anatomy, Physiology, and Radiology,2 and Microbiology, Pathology, and Parasitology,3 College of Veterinary Medicine, and the Department of Statistics,4 North Carolina State University, Raleigh, North Carolina 27606 Received 17 June 1991/Accepted 11 September 1991 Dogs were experimentally inoculated with Rickettsia rickettsii to characterize the comparative efficacies of chloramphenicol, enrofloxacin, and tetracycline for the treatment of Rocky Mountain spotted fever (RMSF). All three antibiotics were equally effective in abrogating the clinical, hematologic, and vascular indicators of rickettsial infection. Antibiotic treatment for 24 h was sufficient to decrease the rickettsemia to levels below detection by Vero cell culture. Early treatment with all three antibiotics resulted in a similar decrease in antibody titer, but acute and convalescent serum samples taken at appropriate times would have still facilitated an accurate diagnosis of RMSF in all but one dog, which did not seroconvert. We conclude that chloramphenicol, enrofloxacin, and tetracycline are equally efficacious for treating experimental canine RMSF.
Rocky Mountain spotted fever (RMSF), caused by Rickettsia rickettsii, is the most common human rickettsiosis in the United States (5, 15). The pathophysiologic consequences of cytopathic rickettsial injury contribute to similar historical findings and physical abnormalities and nearly identical hematologic, serum biochemical, and pathologic changes in both dogs and people (2, 7, 18). Like the disease in humans, canine RMSF is often associated with severe morbidity and occasional mortality (1, 11). Because of these factors, information generated through experimental studies utilizing dogs may be applicable to both species. We are not aware of any controlled clinical or experimental studies that have compared the efficacies of chloramphenicol, enrofloxacin, and tetracycline for the treatment of RMSF. Clinical experience dictates various opinions among veterinarians and physicians as to the comparative efficacies of chloramphenicol and tetracycline for the treatment of RMSF. Recent epidemiologic findings suggest that mortality is higher when chloramphenicol, rather than tetracycline, is used for the treatment of human RMSF (10). However, this trend may be related to physician preference for chloramphenicol in patients with severe disease. Recent demonstration of in vitro rickettsicidal properties of ciprofloxacin, a broad-spectrum quinolone antibiotic (3, 24, 25), suggests that this class of drugs may be useful in the treatment of RMSF. Quinolones may be of particular benefit in those instances when tetracycline or chloramphenicol use is undesirable. Enrofloxacin (Baytril; Mobay Corporation, Animal Health Division, Shawnee, Kans.), an analog of ciprofloxacin, was selected for study because it has received Food and Drug Administration approval for use in dogs and because it is being used with increasing frequency by veterinarians for treating dogs with febrile illness. The purpose of this study was to compare the efficacies of chloramphenicol, enrofloxacin, and tetracycline in dogs experimentally infected with R. rickettsii.
*
MATERIALS AND METHODS
Study design. Sixteen 13-month-old female beagles were randomly divided into four groups, with four dogs in each group (the nontreated control group and chloramphenicol-, enrofloxacin-, and tetracycline-treated groups). All dogs were seronegative to R. rickettsii by microimmunofluorescence testing, as described previously (22), and were negative for dirofilariae and intestinal parasites. Complete blood counts and serum biochemical analyses were normal prior to infection. R. rickettsu. All four groups were inoculated intradermally in two sites over the pelvis (0.5 ml per site) with a canine isolate of R. rickettsii (strain Domino; inoculum strength, 5 x 104 PFU/ml). Rickettsiae were diluted in sterile brain heart infusion broth for inoculation. Treatment protocol. Beginning on postinoculation (p.i.) day 6, antibiotics were administered to the three treatment groups. The mean group dosages were as follows: chloramphenicol, 29.9 mg/kg of body weight orally at 8-h intervals; enrofloxacin, 3.0 mg/kg orally at 12-h intervals; tetracycline HCI, 31.3 mg/kg orally at 8-h intervals. In all three treatment groups, antibiotics were continued for 7 days. Monitoring. Physical examinations, including rectal temperature measurements, observations related to attitudinal change, and oral and ocular examinations, were conducted in an examiner-blinded fashion twice daily. An attitudinal score was recorded for each dog twice daily, with scoring and criteria as follows: 5, alert, active, and eating; 4, alert, inactive, and eating; 3, depressed, inactive, and anorexic; 2, severely depressed and anorexic; 1, recumbent; and 0, dead. Blood was collected by jugular venipuncture on p.i. days 3, 5, 7, 10, 12, 14, 18, and 21 for complete blood counts, serum biochemical analysis, and serologic testing for R. rickettsii by microimmunofluorescence. Vascular permeability studies. Fluorescein angiography of the ocular fundus was performed prior to inoculation and on p.i. days 3, 5, 8, 10, 12, and 17, using previously described methods (7). The area of sodium fluorescein dye leakage from injured retinal vessels was quantitated by computer-
Corresponding author. 2375
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assisted morphometric analysis, using photographic prints (4 by 5 in. [ca. 10 by 13 cm]) of angiograms taken between 30 and 45 s after sodium fluorescein administration. The total surface area of retinal dye leakage per dog was assigned a numerical value corresponding to a fractional area of an optic nerve (which was assigned a numerical value of 1). Drug analysis and pharmacokinetic studies. On p.i. days 6, 11, and 25, blood samples were obtained by jugular venipuncture at 0, 30, 60, 180, 300, and 480 min for pharmacokinetic characterization of each dog within an antibiotic group. For the enrofloxacin group, the last sample was obtained at 720 rather than 480 min. Chloramphenicol, tetracycline HCl, and enrofloxacin concentrations in serum were determined by high performance liquid chromatography, using previously published methods (27, 31, 32). Rickettsial isolation. Sterile, heparinized venous blood was collected from each dog on p.i. day 3 and from two dogs from each group on p. i. days 5, 7, 10, 12, 14, 18, 21, 25, and 28. A modification of the tissue culture isolation method described by Kaplowitz et al. (17) was used. Whole blood (0.10 ml) was evenly distributed onto a confluent monolayer of Vero cells in each of four glass cell culture tubes (16 by 125 mm) from which the medium had been removed. The inoculated Vero cells were incubated at 34°C for 1 h, after which 2 ml of cell culture medium was added and the tubes were reincubated at 34°C at a 5° angle. On the 9th, 14th, 21st, and 28th days, the tubes were vortexed and centrifuged to pellet the cells and debris. The medium was removed, and the pellet was resuspended in phosphate-buffered saline (pH 7.3) containing 0.5% bovine serum albumin. Slides were prepared, air dried, and acetone fixed before being stained for the presence of rickettsiae by indirect fluorescent antibody and direct fluorescent antibody. If there were no detectable rickettsiae during this time, the cultures were considered negative. Microimmunofluorescence testing. A microimmunofluorescence test was used to evaluate for the presence of antibodies to R. rickettsii (strain Sheila Smith) in dogs. Canine sera were evaluated with fluorescein isothiocyanate goat anti-
canine immunoglobulin G (IgG) (heavy- and light-chainspecific) conjugate. All sera were screened at 1:16 dilutions, and the titers of those reacting were determined to the end point. Positive and negative controls were evaluated with each group. Statistics. Antibiotic treatment effects were analyzed by using multivariate repeated-measures analysis of variance. A separate analysis was performed for each physiological parameter. Linear contrasts within the repeated-measures analysis tested for differences between the control and antibiotic treatments. Profile contrasts were used to detect significant differences in the shape of the parameter response curves among the treatments (16). The nonparametric Wilcoxon's rank sum test was performed on attitudinal scores and on the retinal vascular lesion data because of the nonnormality of the data. RESULTS Clinicopathologic studies. Prior to the initiation of antibiotic treatment, there was no significant difference in mean values for rectal temperature, attitudinal score, platelet number, and the ratio of albumin concentration to total protein concentration in serum among the four groups of dogs. Compared with the nontreated control group, antibiotic treatment initiated on p.i. day 6 caused a rapid decrease in rectal temperature (P = 0.001), an improvement in attitudinal score (P = 0.01), and a cessation of albumin loss (P = 0.02). Although the overall drug effect on platelet numbers did not differ significantly between the control and antibiotic treatments, the shape of the platelet response curve over time differed between the control and antibiotic treatments (P = 0.0004). The platelet number returned to preinoculation levels sooner with the antibiotic treatments than with the control. There was no difference in any of the above parameters among the three treatment groups. Fever (rectal temperature 2 39.6°C) was the first indication of infection. All dogs became febrile between p.i. days 2 and 5. Rectal temperature was 40°C in 12 of 16 dogs on p.i.
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ANTIBIOTIC EFFICACY FOR EXPERIMENTAL RMSF IN DOGS
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day 2. Attitudinal scores began to decrease on p.i. day 3, 4, or 5. Antibiotic treatment was initiated within 24 to 72 h after the attitudinal score decreased from 5. Defervescence occurred in the three treatment groups within 24 h after the initiation of antibiotics (Fig. 1), whereas fever (rectal temperature 2 39.6°C) persisted in all four control dogs through p.i. day 10. One control dog remained febrile until p.i. day 17. Daily attitudinal scores ranged from 5 to 3 in all groups (Fig. 2). The attitudinal score in the three treatment groups returned to a 5 by p.i. day 8, whereas control dogs returned toa5byp.i. day 14.
Vascular permeability. In the three treatment groups, platelet numbers (Fig. 3) increased, and the decline in the ratio of albumin concentration to total protein concentratiqn (Fig. 4) stabilized more rapidly than in the nontreated control group. Prior to the initiation of antibiotic therapy, there were no differences in the total number of retinal vascular lesions or in the area of nontapetal retinal sodium fluorescein dye leakage among the four groups of dogs. Nontreated contrpl dogs developed a pattern of retinal vascular lesions typical of experimental R. rickettsii infection (7), with development of lesions on p.i. days 5 to 8 and progression of lesions over the
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BREITSCHWERDT ET AL.
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Postinoculation Days FIG. 4. Mean ratios of albumin to total protein for the control and treatment the initiation of antibiotics (day 6). next 5 to 7 days p.i. Treated dogs either had rapid resolution of existing retinal vascular lesions or failed to develop retinal foci visible with fluorescein angiography, resulting in a decrease in the total number (P = 0.06) (Fig. 5) and morphometric severity (Fig. 6) (P = 0.03) of retinal vascular lesions when compared with control groups. There was no difference in retinal vascular permeability among the three treatment groups as determined by these indicators. Pharmacokinetic studies. Peak concentrations of all three drugs in serum were not affected by R. rickettsii infection. Mean peak concentrations of tetracycline, chloramphenicol,
groups after inoculation with R.
rickettsii (day 0) and after
and enrofloxacin in serum ranged from 2.4 to 3.2 pug/ml, 16.0 to 18.5 ,ug/ml, and 0.48 to 0.81 ,ug/ml, respectively. All concentrations were considered to be in each drug's thera-
peutic range. Rickettsial isolation. Rickettsemia was documented by Vero cell culture in three of the four control dogs but was not detected in any of the treated dogs. Positive rickettsial cultures were obtained only from dogs that were febrile for more than 6 days. None of the treated dogs were febrile for more than 4 days prior to the initiation of antibiotics.
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ANTIBIOTIC EFFICACY FOR EXPERIMENTAL RMSF IN DOGS
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Postinoculation Days Fractional area of optic nerve FIG. 6. Mean surface area of retinal fluorescein leakage in the control and treatment groups after inoculation with R. rickettsii (day 0) and after the initiation of antibiotics (day 6). A numerical value for the lesion surface area was determined by measuring the morphometric area of the lesions compared with the fractional area of the optic nerve.
Positive cultures were obtained from the three control dogs until 24 h prior to defervescence. Serologic results. With the exception of one dog in the enrofloxacin group, which did not seroconvert, IgM and IgG antibodies to R. rickettsii were detected in all dogs (Fig. 7). When the nonseroconverting dog was excluded from the statistical analysis, the serologic response of the control group differed significantly (P = 0.006) from that of the three treatment groups.
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Because reports of therapeutic trials for treating RMSF in dogs or people are lacking, we undertook this study to compare the in vivo efficacies of chloramphenicol, enrofloxacin, and tetracycline. As a result of earlier experimental efficacy studies utilizing chick embryos and mice (21, 28), the clinical use of tetracyclines or chloramphenicol for treating rickettsiosis or ehrlichiosis has become a universally
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BREITSCHWERDT ET AL.
accepted practice (13, 26, 30). Since there are contraindications for both antibiotics that make them less than ideal in some situations, alternative antibiotics for treating rickettsial infections are needed. Recent in vitro (3, 24, 25) and in vivo mouse studies (20), as well as clinical studies (9, 12, 23, 29), indicate that rickettsiae, including R. conorii, R. rickettsii, R. tsutsugamushi, R. typhi, and Ehrlichia sennetsu, are susceptible to quinolone antibiotics such as enrofloxacin or ciprofloxacin. Because enrofloxacin possesses broad-spectrum bactericidal activity against canine pathogens (34), it may prove useful for the treatment of undiagnosed, acute-febrile illness in adult dogs in endemic areas of tick-transmitted rickettsial disease. Our results, in conjunction with recent findings (12, 23-26), also suggest that investigation of the use of a quinolone antibiotic for treating RMSF in people is warranted, particularly in those instances when tetracycline or chloramphenicol use is undesirable. In this study, all three antibiotics were equally effective in abrogating clinical, hematologic, and vascular indicators of rickettsial infection in dogs. Sequential changes in platelet numbers, the ratio of albumin concentration to total protein concentration in serum, and the severity of retinal vascular lesions as determined by fluorescein dye leakage provided the best indicators of rickettsia-induced vascular injury (Fig. 3 to 6). Compared with the more protracted clinical and hematologic course in the control group, treatment with chloramphenicol, enrofloxacin, and tetracycline facilitated, to a similar degree, the cessation of rickettsia-induced vascular injury. This is evidenced by a rapid increase in platelet numbers and a stabilization of the decline in the ratio of albumin concentration to total protein concentration. These indicators coincided with rapid amelioration of focal alterations in retinal vascular permeability after antibiotic therapy or with abrogation of vascular endothelial injury in treated dogs that had not developed lesions. The rapid restoration of functional retinal vascular integrity in these dogs following antibiotic therapy supports previous histopathologic findings in tetracycline-treated guinea pigs with RMSF, which demonstrated rapid changes in endothelial fine structure indicative of cellular repair within 48 to 72 h of the initiation of antibiotic therapy (33). Presumably, the continued decline in the ratio of albumin concentration to total protein concentration in the control dogs reflects the leakage of albumin through damaged blood vessels, with concurrent retention of high-molecular-weight proteins within the vessels. We were not able to detect quantitative differences in attitudinal improvement among the three treatment groups. A study that compared a 2-day course of oral ciprofloxacin and a 2-day course of oral doxycycline for treating Mediterranean spotted fever in people found that patients treated with doxycycline had a shorter defervescence period and a slightly more rapid disappearance of some clinical symptoms (12). Compared with doxycycline, chloramphenicol induces a slower rate of killing of intracellular R. rickettsii in vitro (35), and terramycin induced more consistent death-delaying activity in R. rickettsii-infected embryos than did chloramphenicol (21). Collectively, these studies suggest only minor therapeutic differences between tetracyclines, quinolones, and chloramphenicol. Previous reports on children (14) and dogs (2) suggest that early treatment with antibiotics will decrease the antibody titer to R. rickettsii. In this study, all three antibiotics caused a similar decrease in antibody titer compared with titers for nontreated control dogs. Although antibody titers for the
ANTIMICROB. AGENTS CHEMOTHER.
treated dogs were significantly lower than for the controls, serologic testing of acute and convalescent serum samples taken at appropriate times would have resulted in a fourfold increase in the microimmunofluorescence antibody titers of all but one treated dog, thereby facilitating an accurate diagnosis of RMSF. These results are comparable to a human study in which serodiagnosis (fourfold increase in antibody titer) was possible for six unvaccinated volunteers who developed RMSF and were treated with tetracycline within 6 h after the onset of fever (6). For the first time in the course of our experimental studies related to canine RMSF, a dog that developed mild clinical and hematologic indications of rickettsial infection failed to seroconvert to R. rickettsii by microimmunofluorescence testing. Although the importance of this occurrence to the natural disease state is unclear, it does suggest that early antibiotic administration, or perhaps other factors, might eliminate a measurable serologic response to the organism. In this study, treatment was started on p.i. day 6, compared with an initiation of treatment on day 12 in a previous study (2) that failed to identify a difference in serologic response among tetracycline-treated and nontreated dogs during the same observation period. On the basis of existing information, we conclude that antibiotic treatment for RMSF in people or dogs rarely interferes with serodiagnosis. Rickettsemia was documented only in blood samples obtained between p.i. days 7 and 10 and only from three of the four control dogs. In previous experimental studies with dogs, rickettsemia was detected by tissue culture isolation as early as the first 24 to 48 h of fever, and detectable levels of rickettsemia decrease with defervescence (2, 4, 8). Rickettsiae can be successfully isolated from the blood of febrile human patients if cultures are initiated within the first 36 h of treatment with tetracycline or 48 h with chloramphenicol (8, 17, 19). In the guinea pig model, defervescence occurred after 4 days of tetracycline treatment, but rickettsial organisms could not be found in tissues by immunofluorescent staining after 72 h (33). In our study, treatment with antibiotics resulted in defervescence within 24 h. Our results seem to indicate that the level of rickettsemia was too low to afford positive cultures on p.i. day 3, but other factors, such as the fact that the blood sample was frozen prior to tissue culture inoculation, may have contributed to negative cultures in eight dogs (two dogs from each group) on p.i. day 5. We have been able to demonstrate rickettsemia in dogs on p.i. day 5 by inoculating a Dermacentor variabilis cell culture with fresh plasma (2). It also appears that antibiotic treatment for 24 h was sufficient to lower the number of organisms in the blood to levels below detection. The results of this study must be interpreted with the qualification that infective dose, the strain of R. rickettsii, or potentially other factors might alter the severity of disease and thereby potentially modify the outcome associated with antibiotic treatment. This particular caution in interpretation is emphasized because the case-fatality rate with human RMSF patients treated with chloramphenicol is not significantly lower than with untreated patients (10). Our study may not address the comparative efficacy of these antibiotics in severe, potentially fatal RMSF. REFERENCES 1. Breitschwerdt, E. B., D. J. Meuten, D. H. Walker, M. G. Levy, K. Kennedy, M. King, and B. C. Curtis. 1985. Canine Rocky
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Mountain spotted fever: a kennel epizootic. Am. J. Vet. Res. 46:2124-2128. 2. Breitschwerdt, E. B., D. H. Walker, M. G. Levy, W. Burgdorfer, W. T. Corbett, S. A. Huribert, M. E. Stebbins, B. C. Curtis, and D. A. Alien. 1988. Clinical, hematologic, and humoral immune response in female dogs inoculated with Rickettsia rickettsii and Rickettsia montana. Am. J. Vet. Res. 49:70-76. 3. Broqui, P., and D. Raoult. 1990. In vitro susceptibility of Ehrlichia sennetsu to antibiotics. Antimicrob. Agents Chemother. 34:1593-1596. 4. Buhles, W. C., D. L. Huxsoll, G. Ruch, R. H. Kenyon, and B. L. Elisberg. 1975. Evaluation of primary blood monocyte and bone marrow culture for the isolation of Rickettsia rickettsii. Infect. Immun. 12:1457-1463. 5. Centers for Disease Control. 1983. Annual summary 1982: reported morbidity and mortality in the United States. Morbid. Mortal. Weekly Rep. 31:142. 6. Clements, M. L., J. S. Dumler, P. Fiset, C. L. Wisseman, Jr., M. J. Snyder, and-M. M. Levine. 1983. Serodiagnosis of Rocky Mountain spotted fever: comparison of IgM and IgG enzymelinked immunoabsorbent assays and indirect fluorescent antibody test. J. Infect. Dis. 148:876-880. 7. Davidson, M. G., E. B. Breitschwerdt, D. H. Walker, M. G. Levy, C. S. Carlson, E. M. Hardie, C. A. Grindem, and M. P. Nasisse. 1990. Vascular permeability and coagulation during Rickettsia rickettsii infection in dogs. Am. J. Vet. Res. 51:165170. 8. Davis, J. P., C. M. Wilfert, D. J. Sexton, W. Burgdorfer, E. A. Casper, and R. N. Philip. 1981. Serologic comparison of R. rickettsii isolated from patients in North Carolina to R. rickettsii isolated from patients in Montana, p. 139-147. In W. Burgdorfer and R. L. Anacker (ed.), Rickettsiae and rickettsial diseases. Academic Press, Inc., New York. 9. Eaton, M., M. T. Cohen, D. R. Shlim, and B. Innes. 1989. Ciprofloxin treatment of typhus. JAMA 262:772-773. (Letter.) 10. Fishbein, D. B., M. G. Frontini, R. Giles, and L. L. Vernon. 1990. Fatal cases of Rocky Mountain spotted fever in the United States, 1981-1988. Ann. N.Y. Acad. Sci. 590:246-247. 11. Greene, C. E., W. Burgdorfer, R. Cavagnolo, R. N. Philip, and M. G. Peacock. 1985. Rocky Mountain spotted fever in dogs and its differentiation from canine ehrlichiosis. J. Am. Vet. Med. Assoc. 186:465-472. 12. Gudiol, F., R. Paliares, J. Carratala, F. Bolao, J. Ariza, G. Rufi, and P. F. Viladrich. 1989. Randomized double-blind evaluation of ciprofloxacin and doxycycline for Mediterranean spotted fever. Antimicrob. Agents Chemother. 33:987-988. 13. Hattwick, M. A. W., R. J. L. O'Brien, and B. F. Hanson. 1976. Rocky Mountain spotted fever: epidemiology of an increasing problem. Ann. Intern. Med. 84:732-739. 14. Hays, P. L. 1985. Rocky Mountain spotted fever in children in Kansas: the diagnostic value of an IgM-specific immunofluorescence assay. J. Infect. Dis. 151:369-370. 15. Helmick, C. G., K. W. Bernard, and L. J. D'Angelo. 1984. Rocky Mountain spotted fever: clinical, laboratory, and epidemiological features of 262 cases. J. Infect. Dis. 150:480-488. 16. Johnson, T. A., and D. W. Wichern. 1988. Applied multivariate statistical analysis, 2nd ed., p. 607. Prentice-Hall, Inc., Englewood Cliffs, N.J. 17. Kaplowitz, L. G., J. V. Lange, J. J. Fischer, and D. H. Walker. 1983. Correlation of rickettsial titers, circulating endotoxin and clinical features in Rocky Mountain spotted fever. Arch. Intern. Med. 143:1149-1151. 18. Keenan, K. P., W. C. Buhles, D. L. Huxsol, R. G. Wlliams, P. K. Hildebrandt, J. M. Campbell, and E. H. Stephenson. 1977. Pathogenesis of infection of Rickettsia rickettsii in the dog: a
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disease model for Rocky Mountain spotted fever. J. Infect. Dis. 135:911-917. 19. Marrero, M., and D. Raoult. 1989. Centrifugation-shell vial technique for rapid detection of Mediterranean spotted fever rickettsiae in blood culture. Am. J. Trop. Med. Hyg. 40:197199. 20. McClain, J. B., B. Joshi, and R. Rice. 1988. Chloramphenicol, gentamicin, and ciprofloxacin against murine scrub typhus. Antimicrob. Agents Chemother. 32:285-286. 21. Ormsbee, R. A., H. Parker, and E. G. Pickens. 1955. The comparative effectiveness of aureomycin, terramycin, chloramphenicol, erythromycin, and thiomycetin in suppressing experimental rickettsial infections in chick embryos. J. Infect. Dis. 96:162-167. 22. Philip, R. N., E. A. Casper, R. A. Ormsbee, M. G. Peacock, and W. Burgdorfer. 1976. Microimmunofluorescence test for the serologic study of Rocky Mountain spotted fever and typhus. J. Clin. Microbiol. 3:51-61. 23. Raoult, D., H. Gallais, P. De Micco, and P. Casanova. 1986. Ciprofloxacin therapy for Mediterranean spotted fever. Antimicrob. Agents Chemother. 30:606-607. 24. Raoult, D., P. Roussellier, V. Galicher, R. Perez, and J. Tamalet. 1986. In vitro susceptibility of Rickettsia conorii to ciprofloxacin as determined by suppressing lethality in chicken embryos and by plaque assay. Antimicrob. Agents Chemother. 29:424-425. 25. Raoult, D., P. Roussellier, V. Galicher, and J. Tamalet. 1987. In vitro antibiotic susceptibility of Rickettsia rickettsii and Rickettsia conorii: plaque assay and microplaque colorimetric assay. J. Infect. Dis. 155:1059-1061. 26. Raoult, D., and D. H. Walker. 1990. Rickettsia rickettsii and other spotted fever group rickettsiae (Rocky Mountain spotted fever and other spotted fevers), p. 1465-1471. In G. Mandell, R. G. Douglas, and J. E. Bennett (ed.), Principles and practice of infectious diseases, 3rd ed. Churchill Livingstone, New York. 27. Riond, J. L., and J. E. Riviere. 1989. Doxycycline binding to plasma albumin of several species. J. Vet. Pharmacol. Ther. 12:253-260. 28. Smadel, J. E., E. B. Jackson, and A. B. Cruise. 1948. Chloromycetin in experimental infections. J. Immunol. 62:49-55. 29. Strand, O., and A. Stromberg. 1990. Ciprofloxacin treatment of murine typhus. Scand. J. Infect. Dis. 22:503-504. 30. Troy, G. C., and S. D. Forrester. 1990. Canine ehrlichiosis, p. 404-418. In C. E. Greene (ed.), Infectious diseases of the dog and cat. W. B. Saunders, Philadelphia. 31. Tyczkowska, K. L., K. M. Hedeen, D. P. Aucoin, and A. L. Aronson. 1989. High performance liquid chromatography method for the simultaneous determination of enrofloxacin and its primary metabolite, ciprofloxacin, in canine serum and prostatic tissue. J. Chromatogr. 493:337-346. 32. Tyczkowska, K. L., K. M. Hedeen, D. P. Aucoin, and A. L. Aronson. 1988. Simple LC method for determination of chloramphenicol in equine, canine, and feline serum. J. Chromatogr. Sci. 26:533-536. 33. Walker, D. H., B. G. Cain, J. V. Lange, and A. Harrison. 1981. Pathologic and rickettsial events in tetracycline-treated experimental Rocky Mountain spotted fever, p. 347-362. In W. Burgdorfer, and R. L. Anacker (ed.), Rickettsiae and rickettsial diseases. Academic Press, Inc., New York. 34. Walker, R. D., G. E. Stein, J. G. Hauptman, K. H. MacDonald, S. C. Budsberg, and E. J. Rosser, Jr. 1990. Serum and tissue cage fluid concentrations of ciprofloxacin after oral administration of the drug to healthy dogs. Am. J. Vet. Res. 51:896-900. 35. Wisseman, C. L., and S. V. Ordonez. 1981. Actions of antibiotics on Rickettsia rickettsii. J. Infect. Dis. 153:626-627.
AND
CHEMOTHERAPY, Nov. 1991,
p.
Vol. 35, No. 11
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0066-4804/91/112375-07$02.00/0 Copyright © 1991, American Society for Microbiology
Efficacy of Chloramphenicol, Enrofloxacin, and Tetracycline for Treatment of Experimental Rocky Mountain Spotted Fever in Dogs EDWARD B. BREITSCHWERDT,1* MICHAEL G. DAVIDSON,' DAVID P. AUCOIN,2 MICHAEL G. LEVY,3 NANCY S. SZABADOS,1 BARBARA C. HEGARTY,1 ANITA L. KUEHNE,l AND ROBERT L. JAMES4 Departments of Companion Animal and Special Species Medicine,' Anatomy, Physiology, and Radiology,2 and Microbiology, Pathology, and Parasitology,3 College of Veterinary Medicine, and the Department of Statistics,4 North Carolina State University, Raleigh, North Carolina 27606 Received 17 June 1991/Accepted 11 September 1991 Dogs were experimentally inoculated with Rickettsia rickettsii to characterize the comparative efficacies of chloramphenicol, enrofloxacin, and tetracycline for the treatment of Rocky Mountain spotted fever (RMSF). All three antibiotics were equally effective in abrogating the clinical, hematologic, and vascular indicators of rickettsial infection. Antibiotic treatment for 24 h was sufficient to decrease the rickettsemia to levels below detection by Vero cell culture. Early treatment with all three antibiotics resulted in a similar decrease in antibody titer, but acute and convalescent serum samples taken at appropriate times would have still facilitated an accurate diagnosis of RMSF in all but one dog, which did not seroconvert. We conclude that chloramphenicol, enrofloxacin, and tetracycline are equally efficacious for treating experimental canine RMSF.
Rocky Mountain spotted fever (RMSF), caused by Rickettsia rickettsii, is the most common human rickettsiosis in the United States (5, 15). The pathophysiologic consequences of cytopathic rickettsial injury contribute to similar historical findings and physical abnormalities and nearly identical hematologic, serum biochemical, and pathologic changes in both dogs and people (2, 7, 18). Like the disease in humans, canine RMSF is often associated with severe morbidity and occasional mortality (1, 11). Because of these factors, information generated through experimental studies utilizing dogs may be applicable to both species. We are not aware of any controlled clinical or experimental studies that have compared the efficacies of chloramphenicol, enrofloxacin, and tetracycline for the treatment of RMSF. Clinical experience dictates various opinions among veterinarians and physicians as to the comparative efficacies of chloramphenicol and tetracycline for the treatment of RMSF. Recent epidemiologic findings suggest that mortality is higher when chloramphenicol, rather than tetracycline, is used for the treatment of human RMSF (10). However, this trend may be related to physician preference for chloramphenicol in patients with severe disease. Recent demonstration of in vitro rickettsicidal properties of ciprofloxacin, a broad-spectrum quinolone antibiotic (3, 24, 25), suggests that this class of drugs may be useful in the treatment of RMSF. Quinolones may be of particular benefit in those instances when tetracycline or chloramphenicol use is undesirable. Enrofloxacin (Baytril; Mobay Corporation, Animal Health Division, Shawnee, Kans.), an analog of ciprofloxacin, was selected for study because it has received Food and Drug Administration approval for use in dogs and because it is being used with increasing frequency by veterinarians for treating dogs with febrile illness. The purpose of this study was to compare the efficacies of chloramphenicol, enrofloxacin, and tetracycline in dogs experimentally infected with R. rickettsii.
*
MATERIALS AND METHODS
Study design. Sixteen 13-month-old female beagles were randomly divided into four groups, with four dogs in each group (the nontreated control group and chloramphenicol-, enrofloxacin-, and tetracycline-treated groups). All dogs were seronegative to R. rickettsii by microimmunofluorescence testing, as described previously (22), and were negative for dirofilariae and intestinal parasites. Complete blood counts and serum biochemical analyses were normal prior to infection. R. rickettsu. All four groups were inoculated intradermally in two sites over the pelvis (0.5 ml per site) with a canine isolate of R. rickettsii (strain Domino; inoculum strength, 5 x 104 PFU/ml). Rickettsiae were diluted in sterile brain heart infusion broth for inoculation. Treatment protocol. Beginning on postinoculation (p.i.) day 6, antibiotics were administered to the three treatment groups. The mean group dosages were as follows: chloramphenicol, 29.9 mg/kg of body weight orally at 8-h intervals; enrofloxacin, 3.0 mg/kg orally at 12-h intervals; tetracycline HCI, 31.3 mg/kg orally at 8-h intervals. In all three treatment groups, antibiotics were continued for 7 days. Monitoring. Physical examinations, including rectal temperature measurements, observations related to attitudinal change, and oral and ocular examinations, were conducted in an examiner-blinded fashion twice daily. An attitudinal score was recorded for each dog twice daily, with scoring and criteria as follows: 5, alert, active, and eating; 4, alert, inactive, and eating; 3, depressed, inactive, and anorexic; 2, severely depressed and anorexic; 1, recumbent; and 0, dead. Blood was collected by jugular venipuncture on p.i. days 3, 5, 7, 10, 12, 14, 18, and 21 for complete blood counts, serum biochemical analysis, and serologic testing for R. rickettsii by microimmunofluorescence. Vascular permeability studies. Fluorescein angiography of the ocular fundus was performed prior to inoculation and on p.i. days 3, 5, 8, 10, 12, and 17, using previously described methods (7). The area of sodium fluorescein dye leakage from injured retinal vessels was quantitated by computer-
Corresponding author. 2375
2376
BREITSCHWERDT ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
assisted morphometric analysis, using photographic prints (4 by 5 in. [ca. 10 by 13 cm]) of angiograms taken between 30 and 45 s after sodium fluorescein administration. The total surface area of retinal dye leakage per dog was assigned a numerical value corresponding to a fractional area of an optic nerve (which was assigned a numerical value of 1). Drug analysis and pharmacokinetic studies. On p.i. days 6, 11, and 25, blood samples were obtained by jugular venipuncture at 0, 30, 60, 180, 300, and 480 min for pharmacokinetic characterization of each dog within an antibiotic group. For the enrofloxacin group, the last sample was obtained at 720 rather than 480 min. Chloramphenicol, tetracycline HCl, and enrofloxacin concentrations in serum were determined by high performance liquid chromatography, using previously published methods (27, 31, 32). Rickettsial isolation. Sterile, heparinized venous blood was collected from each dog on p.i. day 3 and from two dogs from each group on p. i. days 5, 7, 10, 12, 14, 18, 21, 25, and 28. A modification of the tissue culture isolation method described by Kaplowitz et al. (17) was used. Whole blood (0.10 ml) was evenly distributed onto a confluent monolayer of Vero cells in each of four glass cell culture tubes (16 by 125 mm) from which the medium had been removed. The inoculated Vero cells were incubated at 34°C for 1 h, after which 2 ml of cell culture medium was added and the tubes were reincubated at 34°C at a 5° angle. On the 9th, 14th, 21st, and 28th days, the tubes were vortexed and centrifuged to pellet the cells and debris. The medium was removed, and the pellet was resuspended in phosphate-buffered saline (pH 7.3) containing 0.5% bovine serum albumin. Slides were prepared, air dried, and acetone fixed before being stained for the presence of rickettsiae by indirect fluorescent antibody and direct fluorescent antibody. If there were no detectable rickettsiae during this time, the cultures were considered negative. Microimmunofluorescence testing. A microimmunofluorescence test was used to evaluate for the presence of antibodies to R. rickettsii (strain Sheila Smith) in dogs. Canine sera were evaluated with fluorescein isothiocyanate goat anti-
canine immunoglobulin G (IgG) (heavy- and light-chainspecific) conjugate. All sera were screened at 1:16 dilutions, and the titers of those reacting were determined to the end point. Positive and negative controls were evaluated with each group. Statistics. Antibiotic treatment effects were analyzed by using multivariate repeated-measures analysis of variance. A separate analysis was performed for each physiological parameter. Linear contrasts within the repeated-measures analysis tested for differences between the control and antibiotic treatments. Profile contrasts were used to detect significant differences in the shape of the parameter response curves among the treatments (16). The nonparametric Wilcoxon's rank sum test was performed on attitudinal scores and on the retinal vascular lesion data because of the nonnormality of the data. RESULTS Clinicopathologic studies. Prior to the initiation of antibiotic treatment, there was no significant difference in mean values for rectal temperature, attitudinal score, platelet number, and the ratio of albumin concentration to total protein concentration in serum among the four groups of dogs. Compared with the nontreated control group, antibiotic treatment initiated on p.i. day 6 caused a rapid decrease in rectal temperature (P = 0.001), an improvement in attitudinal score (P = 0.01), and a cessation of albumin loss (P = 0.02). Although the overall drug effect on platelet numbers did not differ significantly between the control and antibiotic treatments, the shape of the platelet response curve over time differed between the control and antibiotic treatments (P = 0.0004). The platelet number returned to preinoculation levels sooner with the antibiotic treatments than with the control. There was no difference in any of the above parameters among the three treatment groups. Fever (rectal temperature 2 39.6°C) was the first indication of infection. All dogs became febrile between p.i. days 2 and 5. Rectal temperature was 40°C in 12 of 16 dogs on p.i.
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FIG. 1. Mean daily rectal temperature curves foliowing inoculation with R. rickettsii (day 0) and after the initiation of antibiotics (day 6) in the three treatment groups. Each datum point represents the mean value of an AM and PM temperature for four dogs.
ANTIBIOTIC EFFICACY FOR EXPERIMENTAL RMSF IN DOGS
VOL. 35, 1991
2377
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day 2. Attitudinal scores began to decrease on p.i. day 3, 4, or 5. Antibiotic treatment was initiated within 24 to 72 h after the attitudinal score decreased from 5. Defervescence occurred in the three treatment groups within 24 h after the initiation of antibiotics (Fig. 1), whereas fever (rectal temperature 2 39.6°C) persisted in all four control dogs through p.i. day 10. One control dog remained febrile until p.i. day 17. Daily attitudinal scores ranged from 5 to 3 in all groups (Fig. 2). The attitudinal score in the three treatment groups returned to a 5 by p.i. day 8, whereas control dogs returned toa5byp.i. day 14.
Vascular permeability. In the three treatment groups, platelet numbers (Fig. 3) increased, and the decline in the ratio of albumin concentration to total protein concentratiqn (Fig. 4) stabilized more rapidly than in the nontreated control group. Prior to the initiation of antibiotic therapy, there were no differences in the total number of retinal vascular lesions or in the area of nontapetal retinal sodium fluorescein dye leakage among the four groups of dogs. Nontreated contrpl dogs developed a pattern of retinal vascular lesions typical of experimental R. rickettsii infection (7), with development of lesions on p.i. days 5 to 8 and progression of lesions over the
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2378
BREITSCHWERDT ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
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Postinoculation Days FIG. 4. Mean ratios of albumin to total protein for the control and treatment the initiation of antibiotics (day 6). next 5 to 7 days p.i. Treated dogs either had rapid resolution of existing retinal vascular lesions or failed to develop retinal foci visible with fluorescein angiography, resulting in a decrease in the total number (P = 0.06) (Fig. 5) and morphometric severity (Fig. 6) (P = 0.03) of retinal vascular lesions when compared with control groups. There was no difference in retinal vascular permeability among the three treatment groups as determined by these indicators. Pharmacokinetic studies. Peak concentrations of all three drugs in serum were not affected by R. rickettsii infection. Mean peak concentrations of tetracycline, chloramphenicol,
groups after inoculation with R.
rickettsii (day 0) and after
and enrofloxacin in serum ranged from 2.4 to 3.2 pug/ml, 16.0 to 18.5 ,ug/ml, and 0.48 to 0.81 ,ug/ml, respectively. All concentrations were considered to be in each drug's thera-
peutic range. Rickettsial isolation. Rickettsemia was documented by Vero cell culture in three of the four control dogs but was not detected in any of the treated dogs. Positive rickettsial cultures were obtained only from dogs that were febrile for more than 6 days. None of the treated dogs were febrile for more than 4 days prior to the initiation of antibiotics.
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ANTIBIOTIC EFFICACY FOR EXPERIMENTAL RMSF IN DOGS
VOL. 35, 1991
2379
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Postinoculation Days Fractional area of optic nerve FIG. 6. Mean surface area of retinal fluorescein leakage in the control and treatment groups after inoculation with R. rickettsii (day 0) and after the initiation of antibiotics (day 6). A numerical value for the lesion surface area was determined by measuring the morphometric area of the lesions compared with the fractional area of the optic nerve.
Positive cultures were obtained from the three control dogs until 24 h prior to defervescence. Serologic results. With the exception of one dog in the enrofloxacin group, which did not seroconvert, IgM and IgG antibodies to R. rickettsii were detected in all dogs (Fig. 7). When the nonseroconverting dog was excluded from the statistical analysis, the serologic response of the control group differed significantly (P = 0.006) from that of the three treatment groups.
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Because reports of therapeutic trials for treating RMSF in dogs or people are lacking, we undertook this study to compare the in vivo efficacies of chloramphenicol, enrofloxacin, and tetracycline. As a result of earlier experimental efficacy studies utilizing chick embryos and mice (21, 28), the clinical use of tetracyclines or chloramphenicol for treating rickettsiosis or ehrlichiosis has become a universally
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DISCUSSION
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2380
BREITSCHWERDT ET AL.
accepted practice (13, 26, 30). Since there are contraindications for both antibiotics that make them less than ideal in some situations, alternative antibiotics for treating rickettsial infections are needed. Recent in vitro (3, 24, 25) and in vivo mouse studies (20), as well as clinical studies (9, 12, 23, 29), indicate that rickettsiae, including R. conorii, R. rickettsii, R. tsutsugamushi, R. typhi, and Ehrlichia sennetsu, are susceptible to quinolone antibiotics such as enrofloxacin or ciprofloxacin. Because enrofloxacin possesses broad-spectrum bactericidal activity against canine pathogens (34), it may prove useful for the treatment of undiagnosed, acute-febrile illness in adult dogs in endemic areas of tick-transmitted rickettsial disease. Our results, in conjunction with recent findings (12, 23-26), also suggest that investigation of the use of a quinolone antibiotic for treating RMSF in people is warranted, particularly in those instances when tetracycline or chloramphenicol use is undesirable. In this study, all three antibiotics were equally effective in abrogating clinical, hematologic, and vascular indicators of rickettsial infection in dogs. Sequential changes in platelet numbers, the ratio of albumin concentration to total protein concentration in serum, and the severity of retinal vascular lesions as determined by fluorescein dye leakage provided the best indicators of rickettsia-induced vascular injury (Fig. 3 to 6). Compared with the more protracted clinical and hematologic course in the control group, treatment with chloramphenicol, enrofloxacin, and tetracycline facilitated, to a similar degree, the cessation of rickettsia-induced vascular injury. This is evidenced by a rapid increase in platelet numbers and a stabilization of the decline in the ratio of albumin concentration to total protein concentration. These indicators coincided with rapid amelioration of focal alterations in retinal vascular permeability after antibiotic therapy or with abrogation of vascular endothelial injury in treated dogs that had not developed lesions. The rapid restoration of functional retinal vascular integrity in these dogs following antibiotic therapy supports previous histopathologic findings in tetracycline-treated guinea pigs with RMSF, which demonstrated rapid changes in endothelial fine structure indicative of cellular repair within 48 to 72 h of the initiation of antibiotic therapy (33). Presumably, the continued decline in the ratio of albumin concentration to total protein concentration in the control dogs reflects the leakage of albumin through damaged blood vessels, with concurrent retention of high-molecular-weight proteins within the vessels. We were not able to detect quantitative differences in attitudinal improvement among the three treatment groups. A study that compared a 2-day course of oral ciprofloxacin and a 2-day course of oral doxycycline for treating Mediterranean spotted fever in people found that patients treated with doxycycline had a shorter defervescence period and a slightly more rapid disappearance of some clinical symptoms (12). Compared with doxycycline, chloramphenicol induces a slower rate of killing of intracellular R. rickettsii in vitro (35), and terramycin induced more consistent death-delaying activity in R. rickettsii-infected embryos than did chloramphenicol (21). Collectively, these studies suggest only minor therapeutic differences between tetracyclines, quinolones, and chloramphenicol. Previous reports on children (14) and dogs (2) suggest that early treatment with antibiotics will decrease the antibody titer to R. rickettsii. In this study, all three antibiotics caused a similar decrease in antibody titer compared with titers for nontreated control dogs. Although antibody titers for the
ANTIMICROB. AGENTS CHEMOTHER.
treated dogs were significantly lower than for the controls, serologic testing of acute and convalescent serum samples taken at appropriate times would have resulted in a fourfold increase in the microimmunofluorescence antibody titers of all but one treated dog, thereby facilitating an accurate diagnosis of RMSF. These results are comparable to a human study in which serodiagnosis (fourfold increase in antibody titer) was possible for six unvaccinated volunteers who developed RMSF and were treated with tetracycline within 6 h after the onset of fever (6). For the first time in the course of our experimental studies related to canine RMSF, a dog that developed mild clinical and hematologic indications of rickettsial infection failed to seroconvert to R. rickettsii by microimmunofluorescence testing. Although the importance of this occurrence to the natural disease state is unclear, it does suggest that early antibiotic administration, or perhaps other factors, might eliminate a measurable serologic response to the organism. In this study, treatment was started on p.i. day 6, compared with an initiation of treatment on day 12 in a previous study (2) that failed to identify a difference in serologic response among tetracycline-treated and nontreated dogs during the same observation period. On the basis of existing information, we conclude that antibiotic treatment for RMSF in people or dogs rarely interferes with serodiagnosis. Rickettsemia was documented only in blood samples obtained between p.i. days 7 and 10 and only from three of the four control dogs. In previous experimental studies with dogs, rickettsemia was detected by tissue culture isolation as early as the first 24 to 48 h of fever, and detectable levels of rickettsemia decrease with defervescence (2, 4, 8). Rickettsiae can be successfully isolated from the blood of febrile human patients if cultures are initiated within the first 36 h of treatment with tetracycline or 48 h with chloramphenicol (8, 17, 19). In the guinea pig model, defervescence occurred after 4 days of tetracycline treatment, but rickettsial organisms could not be found in tissues by immunofluorescent staining after 72 h (33). In our study, treatment with antibiotics resulted in defervescence within 24 h. Our results seem to indicate that the level of rickettsemia was too low to afford positive cultures on p.i. day 3, but other factors, such as the fact that the blood sample was frozen prior to tissue culture inoculation, may have contributed to negative cultures in eight dogs (two dogs from each group) on p.i. day 5. We have been able to demonstrate rickettsemia in dogs on p.i. day 5 by inoculating a Dermacentor variabilis cell culture with fresh plasma (2). It also appears that antibiotic treatment for 24 h was sufficient to lower the number of organisms in the blood to levels below detection. The results of this study must be interpreted with the qualification that infective dose, the strain of R. rickettsii, or potentially other factors might alter the severity of disease and thereby potentially modify the outcome associated with antibiotic treatment. This particular caution in interpretation is emphasized because the case-fatality rate with human RMSF patients treated with chloramphenicol is not significantly lower than with untreated patients (10). Our study may not address the comparative efficacy of these antibiotics in severe, potentially fatal RMSF. REFERENCES 1. Breitschwerdt, E. B., D. J. Meuten, D. H. Walker, M. G. Levy, K. Kennedy, M. King, and B. C. Curtis. 1985. Canine Rocky
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