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Use of enrofloxacin in the treatment of canine brucellosis in a dog kennel (clinical trial)
Theriogenology 66 (2006) 1573–1578 www.journals.elsevierhealth.com/periodicals/the
Use of enrofloxacin in the treatment of canine brucellosis in a dog kennel (clinical trial) M.M. Wanke a,*, M.V. Delpino b, P.C. Baldi b a
Faculty of Veterinary Science, University of Buenos Aires, Theriogenology, Chorroarin 280 (1427), Buenos Aires, Argentina b Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Immunology, Argentina
Abstract To date, no totally effective antibiotic for the eradication of canine brucellosis has been found. The purpose of this study was to evaluate the efficacy of enrofloxacin in a kennel infected with Brucella canis. Twelve dogs, 2 males and 10 females (including 1 in estrus, 3 pregnant, and 6 in anestrus) infected with B. canis were given 5 mg/kg of enrofloxacin orally every 12 h for 30 days. Females received additional courses of enrofloxacin during the estral and luteal phases of the subsequent cycles (0–2 cycles). They were repeatedly mated by infected males. A serological follow-up was carried out for 38 months. The clinical, serological and bacteriological findings were recorded. In a trial carried out 14 months after the beginning of this study, all dogs were negative on the Rapid Slide Agglutination Test (RSAT). No abortions were observed. All mated female dogs conceived and gave birth to healthy puppies. Cultures of postpartum vaginal discharges (lochia) were negative for B. canis. Similar to other treatments, although enrofloxacin was not completely efficacious in treating canine brucellosis, it maintained fertility and avoided the recurrence of abortions, transmission of the disease to the puppies and dissemination of microorganisms during parturition. We inferred that enrofloxacin could be used as an alternative drug for the treatment of canine brucellosis. # 2006 Elsevier Inc. All rights reserved. Keywords: Brucella canis; Canine brucellosis; Enrofloxacin; Antibiotics; Antibodies
1. Introduction Canine brucellosis can cause large economic losses among dog breeders. Because of its intracellular replication, Brucella canis is difficult to eliminate with antibiotics. Several in vitro studies using single or combined antibiotics showed that B. canis is sensitive to tetracyclines, aminoglucosides, chloramphenicol, spectinomycin, rifamicyn, sulfadimethoxine and fluoroquinolones [1,2]. Animals tested in vivo * Corresponding author. Tel.: +54 11 4524 8425; fax: +54 11 4524 8425. E-mail addresses: [email protected], [email protected] (M.M. Wanke). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.01.034
responded well to treatments with either minocycline or tetracycline, when combined with streptomycin [3– 5]. However, none of the treatments proved to be 100% effective in eradicating the disease. Even if bacteria are apparently successfully eliminated from the infected dog, Brucella survives in some tissues, including lymph nodes, spleen, uterus and prostate [6]. Frequently, serum antibodies reappear after the treatment is completed. In female dogs, this happens usually after estrus (in the intact bitch) or after stressful situations. Bitches may conceive and give birth even without antibiotic treatment, but the puppies are born infected, thus spreading the disease to the rest of the kennel. Male dogs usually become sterile due to severe damage to the epididymis and
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testes, but they still have the potential to transmit the disease. Although enrofloxacin has proved to be effective in vitro against B. canis and is recommended by the manufacturers for the treatment of canine brucellosis, no studies have been published regarding its efficacy for this disease. The purpose of this study was to evaluate the efficacy of enrofloxacin treatment for controlling a kennel infected with B. canis. 2. Materials and methods 2.1. Animals and treatments Twenty-nine dogs were included in this study (24 Poodles, 3 Yorkshire terriers, 1 Dachshund, and 1 German Shepherd); all belonged to a kennel where several abortions had been reported. Animals having Brucellosis were isolated from healthy ones, and each group was treated and managed by different personnel. Kennels were cleaned daily with a quaternary ammonia disinfectant. Dogs that became serologically positive after the beginning of the treatment were immediately placed with infected animals. All dogs, both positive and negative, were treated with 5 mg/kg of enrofloxacin orally every 12 h for 30 days. Previous studies have shown that antibiotic therapy during gestation prevented abortions and allowed whelping of healthy litters [7]. Otherwise, estrus vaginal discharges can be a source of environmental spread. Therefore, in the present study, infected females received additional enrofloxacin courses during all subsequent estrual and luteal phases during the course of this study (range, 0–2 cycles). 2.2. Serology and ELISA assays 2.2.1. The 2-mercapto-ethanol Rapid Slide Agglutination Test (2ME-RSAT) The 2ME-RSAT using the less mucoid M( ) strain of B. canis was carried out as described by Carmichael and Joubert [8] to determine agglutinating antibodies against B. canis. Positive reactions were rated from 1+ (slight agglutination, small clumps) to 4+ (complete agglutination, thick clumps). Serum antibodies to HS (hot saline extract) were detected by ELISA, as described previously [9]. B. canis M( ) cells harvested in the logarithmic phase of growth were washed, re-suspended in buffered saline and autoclaved at 125 8C for 30 min. After lowspeed centrifugation, the supernatant was ultracentrifuged and the pellet (HS) was dissolved in distilled
water. Polystyrene plates were coated with HS (0.5 mg of protein/well) and blocked with 3% skim milk. After incubation with serial dilutions of the sera under study, anti-HS antibodies were detected by incubation with an appropriate anti-dog IgG conjugate and o-phenylenediamine/H2O2. The reaction was stopped with 4N H2SO4 and the optical density (OD) was read at 490 nm in a microplate ELISA reader (Metertech Inc.). Serum reactivity against Brucella lumazine synthase (LS) was measured by an indirect ELISA similar to that described above. Recombinant LS, obtained as described previously [10], was adsorbed onto Maxisorp plates at 0.3 mg/ well. Previous studies from our group [9] have shown that this ELISA is useful for the diagnosis of canine brucellosis. ELISA cut-off values were calculated as the mean OD of 50 normal sera, plus three standard deviations. A reactivity index was calculated as the ratio between the OD of the sample and the respective cut-off values. An index >1 indicated a positive result by ELISA. 2.3. Bacteriological studies B. canis was isolated as previously described [11] from blood, semen or vaginal discharge samples. Suspected cultures were subcultured on solid medium for identification at the Instituto Nacional de Microbiologı´a Dr. C.G. Malbra´n. 2.4. Follow-up Dogs were initially tested by 2ME-RSAT and ELISA (HS and LS). Blood and vaginal discharge samples were obtained for bacteriological tests from dogs that tested positive in any serological test. Follow-up antibody titrations were conducted every 2 weeks for the first 1.5 months, then monthly for the next 5 months, and finally, after 9.5, 14.5 and 38 months. Bacteriological investigation of B. canis was performed on samples of blood and vaginal discharges obtained after parturitions within 12 months after treatment. Serum samples from three female puppies, which were born in the first post-treatment delivery, were tested 2 months after their first estrus. In all subsequent estrual periods, all previously infected and treated females were mated by the infected (now treated) male dog that had spread the disease into the kennel. All matings, pregnancies, deliveries, rates of perinatal mortality and presence of abnormal secretions of each female dog, were recorded.
M.M. Wanke et al. / Theriogenology 66 (2006) 1573–1578
3. Results From 29 studied dogs, 17 were serologically negative and asymptomatic during the entire study. Initial clinical findings of the 12 infected dogs are presented in Table 1. 3.1. Initial results and appearance of new cases At the beginning of the experiment, 2ME-RSAT was clearly positive in six dogs, weakly positive in two, and negative in four. Two of the latter were positive by ELISA tests. In the sampling carried out 1 month later, dogs initially showing weak agglutination exhibited strong 2ME-RSAT reactions. In addition, the two dogs that were initially negative by agglutination but positive by ELISA, had slight agglutination reactions. One of the two dogs that were initially negative by all the tests became slightly positive by both agglutination and ELISA. A female (no. 4) that tested positive in the initial sampling became negative in the following monthly sampling. By the second month, 10 dogs were positive by agglutination. The remaining initially negative dog (Animal 12) became positive by ELISA at this sampling, and then became positive by 2ME-RSAT in the third month. 3.2. Evolution of antibody levels and response to treatment In general, the three serological tests yielded similar qualitative results, but anti-HS reactivities tended to be higher than anti-LS and 2ME-RSAT values. The evolution of the antibody levels is shown in Fig. 1.
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Apart from the initial treatment, nine females received additional enrofloxacin courses during subsequent estrous periods and pregnancies. In dog no. 4, the agglutination reaction turned negative after 1 month and anti-LS antibodies reached a cut-off value at the 4.5-month sample, but anti-LS reactivity was persistently positive. Both males and six bitches (1, 3, 6, 7, 8 and 9) had similar antibody responses. They turned negative for agglutination at 9.5 or 1.5 months and for anti-LS ELISA at 9.5 months. In general, anti-HS responses were more variable. Both males and four females became negative for anti-HS antibodies within 38 months, whereas two females had persistent reactivity slightly above the cut-off point. In female no. 8, in which the agglutination decline took longer, anti-HS antibodies remained high at the 14.5-month evaluation. Unfortunately, this female died before the end of the trial. At the beginning of the trial, Female 12 had a 5-day pregnancy and was negative by all the tests. Antibiotics were administered to this dog for 2 months, even after delivery. Positive reactions were observed in this animal at 1 month (ELISA) or 2 month (agglutination) after the beginning of the experiment. After 8 months, the female displayed estrus and bred, after which it received a 2.5-month treatment. At 9.5 months, the agglutination test was negative and it remained unchanged until month 38. During that period, this dog received two more enrofloxacin courses. Although ELISA titers declined, they were still positive at month 38. All her subsequent pregnancies were successful, giving birth to healthy litters, and the cultures of postpartum vaginal discharges were negative. During follow-up, Female 11 maintained the same levels of antibodies as those observed at the beginning of the trial. This female was
Table 1 Age, sex, results of the bacteriologic cultures, clinical signs, and agglutination of 12 poodles naturally infected with Brucella canis at the beginning of the study No.
Gender
Age (years)
Culture
Symptoms
2ME-RSAT
1 2 3 4 5 6 7 8 9 10 11 12
F F F F M F F F F M F F
4 3 3 5 2 3 3 3 2 4 5 5
Positive Negative Positive Negative Positive Negative/FV+ Negative Negative Negative Negative Positive Positive
None Perinatal mortality 1 month earlier Abortion None None Pregnant 45 days (aborted at Day 48) Abortion Abortion No No Pregnant 32 days (aborted at Day 50) Pregnant 5 days (normal parturition)
3 3 4 4 2 3 3 0 0 0 0
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Fig. 1. Evolution of antibody levels detected with 2ME-RSAT, ELISA anti LPS and ELISA anti-LS in 12 poodles naturally infected with Brucella canis and treated with enrofloxacin.
never observed in estrus during the experiment and it died after 14 months of follow-up. Finally, Female 2 had a good initial response to the treatment, with declining antibody levels in the first 5 months. At 4
months of follow-up, this dog exhibited estrus and mated. Even though it received the treatment for 2 months, the antibody titers increased during this period. Unfortunately, the female died before the 14.5-month
M.M. Wanke et al. / Theriogenology 66 (2006) 1573–1578
sampling, but based on its immunological response, we inferred that the second treatment was not effective. 3.3. Bacterial cultures Bacteria were isolated from blood in only 5 of 12 dogs, and from postpartum discharges in 1 female whose blood culture had been negative. 3.4. Clinical observations None of the males exhibited orchitis or epididymitis, and their fertility was not affected. At the beginning of the experiment, three females (6, 11 and 12) were pregnant. They were administered antibiotics immediately. Female 6 had a 45-day pregnancy, but aborted 3 days later; Female 11 was 11 days into a pregnancy and aborted on Day 50; Female 12 was 15 days pregnant and gave birth to normal puppies after a full-term pregnancy. The vaginal discharge cultured at whelping was negative. For the remainder of the trial, fertility of all the females was normal; there were no further abortions or stillborn puppies. Cultures of postpartum discharges were all negative. Three female puppies born from infected mothers were serologically tested after puberty and yielded negative results. 4. Discussion In spite of the known in vitro sensitivity of B. canis to enrofloxacin, no clinical trials have been performed to test the efficacy of this drug against canine brucellosis. In this study, enrofloxacin was used to treat an outbreak of canine brucellosis in a kennel. The efficacy of the drug was monitored by means of a clinical and serological follow-up. Clinical results were satisfactory, since enrofloxacin therapy appeared to prevent bacterial dissemination and preserved the fertility of males and females. In most cases, serological results coincided with the clinical outcome of the animals. However, in spite of this favorable clinical response, some dogs had persistent antibody levels. Antibody levels declined slowly after treatment, especially anti-HS antibodies as measured by ELISA. The rough lipopolysaccharide (R-LPS) contained in HS is particularly immunogenic and it is possible that any reappearance of the bacterium in the blood may trigger sufficient anti-R-LPS antibodies to be detected by the ELISA technique. Both males exhibited a reduction of antibody titers at about 4.5 months after initial treatment, and this also was observed in seven females.
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In most of the females, there was an increase in antibody levels before estrus and a decrease after the second treatment. By contrast, in the males, the decline in antibody titers was more continuous and stable. It is unclear why Female 4, which had the best antibody response as measured by 2ME-RSAT and anti-LS ELISA, did not attain negative anti-HS titers. Female 2, in spite of having increased titers after the second treatment, gave birth to healthy puppies and the cultures of its postpartum secretions were negative. Even though the follow-up of this female could not be completed, its serological response led us to speculate that it developed resistance to the antibiotic. The antibody response of Female 12 was surprising. Agglutination titers declined as expected, but anti-LS and anti-HS antibodies remained high, suggesting a persistence of the bacterium. However, all pregnancies were normal and the puppies were healthy. The cultures of postpartum vaginal discharges were negative. The discrepancy between agglutination and ELISA results has been observed previously and could be attributed to the development of non-agglutinating antibodies [12]. The results from the present study confirmed our previous findings showing that, while 2ME-RSAT was useful as screening test in acute cases, ELISA tests had higher diagnostic sensitivity in both acute and chronic phases of the disease [9]. Three of the female puppies born from infected and treated bitches tested negative after reaching puberty, which indicated that their mothers succeeded in breeding without disseminating the disease. Although B. canis was isolated from only six infected dogs, most of the females that yielded negative blood cultures had been treated with antibiotics before sampling, because enrofloxacin was administered in response to the abortion. The two dogs that became serologically positive in the second sampling had also received antimicrobial prophylaxis before the blood culture sampling. While the results obtained with enrofloxacin in this study were not superior to those obtained with other antimicrobial treatments previously tested, this monotherapy offers the advantage of avoiding the need of combination with streptomycin, which has undesirable toxic effects. An additional advantage of enrofloxacin is the possibility of its use during gestation. Although some textbooks list this drug as not recommended during gestation, this limitation is not mentioned by the manufacturers. Notably, the litters born in this study did not show any health problems. In conclusion, enrofloxacin is an alternative drug to be used in the treatment of canine brucellosis. Its
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efficacy was similar to that described for streptomycin combined with tetracycline or minocycline. Acknowledgements Our thanks to Dr. Nidia Lucero from Instituto Nacional de Microbiologı´a and ‘‘Dr. C.G. Malbra´n’’ for carrying out the bacteriological cultures. References [1] Mateu de Antonio EM, Martin M. In vitro efficacy of several antimicrobial combinations against Brucella canis and Brucella melitensis strains isolated from dogs. Vet Microbiol 1995;45: 1–10. [2] Terakado N, Ueda H, Sugawara H, Isayama Y, Koyama N. Drug susceptibility of Brucella canis isolated from dogs. Jpn J Vet Sci 1978;40:291–5. [3] Castro RF, Carmichael LE. Brucella canis infection in dogs: treatment trials. Rev Latinoam Microbiol 1981;23:75–9. [4] Nicoletti P. Further studies on the use of antibiotics in canine brucellosis. Compend Cont Ed Pract Vet 1991;13(944):946–7.
[5] Zoha SJ, Walsh R. Effect of a two-stage antibiotic treatment regimen on dogs naturally infected with Brucella canis. J Am Vet Med Assoc 1982;180:1474–5. [6] Lewis Jr GE, Crumrine MH, Jennings PB, Fariss BL. Therapeutic value of tetracycline and ampicillin in dogs infected with Brucella canis. J Am Vet Med Assoc 1973;163:239–41. [7] Johnson CA, Bennett M, Jensen RK, Schirmer R. Effect of combined antibiotic therapy on fertility in brood bitches infected with Brucella canis. J Am Vet Med Assoc 1982;180:1330–3. [8] Carmichael LE, Joubert JC. A rapid slide agglutination test for the serodiagnosis of Brucella canis infection that employs a variant (M ) organism as antigen. Cornell Vet 1987;77:3–12. [9] Wanke MM, Delpino MV, Baldi PC. Comparative performance of ELISA assay using cytosolic or outer membrane antigens of Brucella for the serodiagnosis of canine brucellosis. Vet Microbiol 2002;88:367–75. [10] Goldbaum FA, Velikovsky CA, Baldi PC, Mo¨rtl S, Bacher A, Fossati CA. The 18 kDa cytoplasmic protein of Brucella spp., an antigen useful for diagnosis, is a lumazine synthase. J Med Microbiol 1999;48:833–9. [11] Carmichael LE, Shin SJ. Canine brucellosis: a diagnostician’s dilemma. Semin Vet Med Surg (Small Anim) 1996;11:161–5. [12] Parma AE, Santisteban G, Margni RA. Analysis and in vivo assay of B. abortus agglutinating and non-agglutinating antibodies. Vet Microbiol 1984;9:391–8.
Use of enrofloxacin in the treatment of canine brucellosis in a dog kennel (clinical trial) M.M. Wanke a,*, M.V. Delpino b, P.C. Baldi b a
Faculty of Veterinary Science, University of Buenos Aires, Theriogenology, Chorroarin 280 (1427), Buenos Aires, Argentina b Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Immunology, Argentina
Abstract To date, no totally effective antibiotic for the eradication of canine brucellosis has been found. The purpose of this study was to evaluate the efficacy of enrofloxacin in a kennel infected with Brucella canis. Twelve dogs, 2 males and 10 females (including 1 in estrus, 3 pregnant, and 6 in anestrus) infected with B. canis were given 5 mg/kg of enrofloxacin orally every 12 h for 30 days. Females received additional courses of enrofloxacin during the estral and luteal phases of the subsequent cycles (0–2 cycles). They were repeatedly mated by infected males. A serological follow-up was carried out for 38 months. The clinical, serological and bacteriological findings were recorded. In a trial carried out 14 months after the beginning of this study, all dogs were negative on the Rapid Slide Agglutination Test (RSAT). No abortions were observed. All mated female dogs conceived and gave birth to healthy puppies. Cultures of postpartum vaginal discharges (lochia) were negative for B. canis. Similar to other treatments, although enrofloxacin was not completely efficacious in treating canine brucellosis, it maintained fertility and avoided the recurrence of abortions, transmission of the disease to the puppies and dissemination of microorganisms during parturition. We inferred that enrofloxacin could be used as an alternative drug for the treatment of canine brucellosis. # 2006 Elsevier Inc. All rights reserved. Keywords: Brucella canis; Canine brucellosis; Enrofloxacin; Antibiotics; Antibodies
1. Introduction Canine brucellosis can cause large economic losses among dog breeders. Because of its intracellular replication, Brucella canis is difficult to eliminate with antibiotics. Several in vitro studies using single or combined antibiotics showed that B. canis is sensitive to tetracyclines, aminoglucosides, chloramphenicol, spectinomycin, rifamicyn, sulfadimethoxine and fluoroquinolones [1,2]. Animals tested in vivo * Corresponding author. Tel.: +54 11 4524 8425; fax: +54 11 4524 8425. E-mail addresses: [email protected], [email protected] (M.M. Wanke). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.01.034
responded well to treatments with either minocycline or tetracycline, when combined with streptomycin [3– 5]. However, none of the treatments proved to be 100% effective in eradicating the disease. Even if bacteria are apparently successfully eliminated from the infected dog, Brucella survives in some tissues, including lymph nodes, spleen, uterus and prostate [6]. Frequently, serum antibodies reappear after the treatment is completed. In female dogs, this happens usually after estrus (in the intact bitch) or after stressful situations. Bitches may conceive and give birth even without antibiotic treatment, but the puppies are born infected, thus spreading the disease to the rest of the kennel. Male dogs usually become sterile due to severe damage to the epididymis and
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testes, but they still have the potential to transmit the disease. Although enrofloxacin has proved to be effective in vitro against B. canis and is recommended by the manufacturers for the treatment of canine brucellosis, no studies have been published regarding its efficacy for this disease. The purpose of this study was to evaluate the efficacy of enrofloxacin treatment for controlling a kennel infected with B. canis. 2. Materials and methods 2.1. Animals and treatments Twenty-nine dogs were included in this study (24 Poodles, 3 Yorkshire terriers, 1 Dachshund, and 1 German Shepherd); all belonged to a kennel where several abortions had been reported. Animals having Brucellosis were isolated from healthy ones, and each group was treated and managed by different personnel. Kennels were cleaned daily with a quaternary ammonia disinfectant. Dogs that became serologically positive after the beginning of the treatment were immediately placed with infected animals. All dogs, both positive and negative, were treated with 5 mg/kg of enrofloxacin orally every 12 h for 30 days. Previous studies have shown that antibiotic therapy during gestation prevented abortions and allowed whelping of healthy litters [7]. Otherwise, estrus vaginal discharges can be a source of environmental spread. Therefore, in the present study, infected females received additional enrofloxacin courses during all subsequent estrual and luteal phases during the course of this study (range, 0–2 cycles). 2.2. Serology and ELISA assays 2.2.1. The 2-mercapto-ethanol Rapid Slide Agglutination Test (2ME-RSAT) The 2ME-RSAT using the less mucoid M( ) strain of B. canis was carried out as described by Carmichael and Joubert [8] to determine agglutinating antibodies against B. canis. Positive reactions were rated from 1+ (slight agglutination, small clumps) to 4+ (complete agglutination, thick clumps). Serum antibodies to HS (hot saline extract) were detected by ELISA, as described previously [9]. B. canis M( ) cells harvested in the logarithmic phase of growth were washed, re-suspended in buffered saline and autoclaved at 125 8C for 30 min. After lowspeed centrifugation, the supernatant was ultracentrifuged and the pellet (HS) was dissolved in distilled
water. Polystyrene plates were coated with HS (0.5 mg of protein/well) and blocked with 3% skim milk. After incubation with serial dilutions of the sera under study, anti-HS antibodies were detected by incubation with an appropriate anti-dog IgG conjugate and o-phenylenediamine/H2O2. The reaction was stopped with 4N H2SO4 and the optical density (OD) was read at 490 nm in a microplate ELISA reader (Metertech Inc.). Serum reactivity against Brucella lumazine synthase (LS) was measured by an indirect ELISA similar to that described above. Recombinant LS, obtained as described previously [10], was adsorbed onto Maxisorp plates at 0.3 mg/ well. Previous studies from our group [9] have shown that this ELISA is useful for the diagnosis of canine brucellosis. ELISA cut-off values were calculated as the mean OD of 50 normal sera, plus three standard deviations. A reactivity index was calculated as the ratio between the OD of the sample and the respective cut-off values. An index >1 indicated a positive result by ELISA. 2.3. Bacteriological studies B. canis was isolated as previously described [11] from blood, semen or vaginal discharge samples. Suspected cultures were subcultured on solid medium for identification at the Instituto Nacional de Microbiologı´a Dr. C.G. Malbra´n. 2.4. Follow-up Dogs were initially tested by 2ME-RSAT and ELISA (HS and LS). Blood and vaginal discharge samples were obtained for bacteriological tests from dogs that tested positive in any serological test. Follow-up antibody titrations were conducted every 2 weeks for the first 1.5 months, then monthly for the next 5 months, and finally, after 9.5, 14.5 and 38 months. Bacteriological investigation of B. canis was performed on samples of blood and vaginal discharges obtained after parturitions within 12 months after treatment. Serum samples from three female puppies, which were born in the first post-treatment delivery, were tested 2 months after their first estrus. In all subsequent estrual periods, all previously infected and treated females were mated by the infected (now treated) male dog that had spread the disease into the kennel. All matings, pregnancies, deliveries, rates of perinatal mortality and presence of abnormal secretions of each female dog, were recorded.
M.M. Wanke et al. / Theriogenology 66 (2006) 1573–1578
3. Results From 29 studied dogs, 17 were serologically negative and asymptomatic during the entire study. Initial clinical findings of the 12 infected dogs are presented in Table 1. 3.1. Initial results and appearance of new cases At the beginning of the experiment, 2ME-RSAT was clearly positive in six dogs, weakly positive in two, and negative in four. Two of the latter were positive by ELISA tests. In the sampling carried out 1 month later, dogs initially showing weak agglutination exhibited strong 2ME-RSAT reactions. In addition, the two dogs that were initially negative by agglutination but positive by ELISA, had slight agglutination reactions. One of the two dogs that were initially negative by all the tests became slightly positive by both agglutination and ELISA. A female (no. 4) that tested positive in the initial sampling became negative in the following monthly sampling. By the second month, 10 dogs were positive by agglutination. The remaining initially negative dog (Animal 12) became positive by ELISA at this sampling, and then became positive by 2ME-RSAT in the third month. 3.2. Evolution of antibody levels and response to treatment In general, the three serological tests yielded similar qualitative results, but anti-HS reactivities tended to be higher than anti-LS and 2ME-RSAT values. The evolution of the antibody levels is shown in Fig. 1.
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Apart from the initial treatment, nine females received additional enrofloxacin courses during subsequent estrous periods and pregnancies. In dog no. 4, the agglutination reaction turned negative after 1 month and anti-LS antibodies reached a cut-off value at the 4.5-month sample, but anti-LS reactivity was persistently positive. Both males and six bitches (1, 3, 6, 7, 8 and 9) had similar antibody responses. They turned negative for agglutination at 9.5 or 1.5 months and for anti-LS ELISA at 9.5 months. In general, anti-HS responses were more variable. Both males and four females became negative for anti-HS antibodies within 38 months, whereas two females had persistent reactivity slightly above the cut-off point. In female no. 8, in which the agglutination decline took longer, anti-HS antibodies remained high at the 14.5-month evaluation. Unfortunately, this female died before the end of the trial. At the beginning of the trial, Female 12 had a 5-day pregnancy and was negative by all the tests. Antibiotics were administered to this dog for 2 months, even after delivery. Positive reactions were observed in this animal at 1 month (ELISA) or 2 month (agglutination) after the beginning of the experiment. After 8 months, the female displayed estrus and bred, after which it received a 2.5-month treatment. At 9.5 months, the agglutination test was negative and it remained unchanged until month 38. During that period, this dog received two more enrofloxacin courses. Although ELISA titers declined, they were still positive at month 38. All her subsequent pregnancies were successful, giving birth to healthy litters, and the cultures of postpartum vaginal discharges were negative. During follow-up, Female 11 maintained the same levels of antibodies as those observed at the beginning of the trial. This female was
Table 1 Age, sex, results of the bacteriologic cultures, clinical signs, and agglutination of 12 poodles naturally infected with Brucella canis at the beginning of the study No.
Gender
Age (years)
Culture
Symptoms
2ME-RSAT
1 2 3 4 5 6 7 8 9 10 11 12
F F F F M F F F F M F F
4 3 3 5 2 3 3 3 2 4 5 5
Positive Negative Positive Negative Positive Negative/FV+ Negative Negative Negative Negative Positive Positive
None Perinatal mortality 1 month earlier Abortion None None Pregnant 45 days (aborted at Day 48) Abortion Abortion No No Pregnant 32 days (aborted at Day 50) Pregnant 5 days (normal parturition)
3 3 4 4 2 3 3 0 0 0 0
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Fig. 1. Evolution of antibody levels detected with 2ME-RSAT, ELISA anti LPS and ELISA anti-LS in 12 poodles naturally infected with Brucella canis and treated with enrofloxacin.
never observed in estrus during the experiment and it died after 14 months of follow-up. Finally, Female 2 had a good initial response to the treatment, with declining antibody levels in the first 5 months. At 4
months of follow-up, this dog exhibited estrus and mated. Even though it received the treatment for 2 months, the antibody titers increased during this period. Unfortunately, the female died before the 14.5-month
M.M. Wanke et al. / Theriogenology 66 (2006) 1573–1578
sampling, but based on its immunological response, we inferred that the second treatment was not effective. 3.3. Bacterial cultures Bacteria were isolated from blood in only 5 of 12 dogs, and from postpartum discharges in 1 female whose blood culture had been negative. 3.4. Clinical observations None of the males exhibited orchitis or epididymitis, and their fertility was not affected. At the beginning of the experiment, three females (6, 11 and 12) were pregnant. They were administered antibiotics immediately. Female 6 had a 45-day pregnancy, but aborted 3 days later; Female 11 was 11 days into a pregnancy and aborted on Day 50; Female 12 was 15 days pregnant and gave birth to normal puppies after a full-term pregnancy. The vaginal discharge cultured at whelping was negative. For the remainder of the trial, fertility of all the females was normal; there were no further abortions or stillborn puppies. Cultures of postpartum discharges were all negative. Three female puppies born from infected mothers were serologically tested after puberty and yielded negative results. 4. Discussion In spite of the known in vitro sensitivity of B. canis to enrofloxacin, no clinical trials have been performed to test the efficacy of this drug against canine brucellosis. In this study, enrofloxacin was used to treat an outbreak of canine brucellosis in a kennel. The efficacy of the drug was monitored by means of a clinical and serological follow-up. Clinical results were satisfactory, since enrofloxacin therapy appeared to prevent bacterial dissemination and preserved the fertility of males and females. In most cases, serological results coincided with the clinical outcome of the animals. However, in spite of this favorable clinical response, some dogs had persistent antibody levels. Antibody levels declined slowly after treatment, especially anti-HS antibodies as measured by ELISA. The rough lipopolysaccharide (R-LPS) contained in HS is particularly immunogenic and it is possible that any reappearance of the bacterium in the blood may trigger sufficient anti-R-LPS antibodies to be detected by the ELISA technique. Both males exhibited a reduction of antibody titers at about 4.5 months after initial treatment, and this also was observed in seven females.
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In most of the females, there was an increase in antibody levels before estrus and a decrease after the second treatment. By contrast, in the males, the decline in antibody titers was more continuous and stable. It is unclear why Female 4, which had the best antibody response as measured by 2ME-RSAT and anti-LS ELISA, did not attain negative anti-HS titers. Female 2, in spite of having increased titers after the second treatment, gave birth to healthy puppies and the cultures of its postpartum secretions were negative. Even though the follow-up of this female could not be completed, its serological response led us to speculate that it developed resistance to the antibiotic. The antibody response of Female 12 was surprising. Agglutination titers declined as expected, but anti-LS and anti-HS antibodies remained high, suggesting a persistence of the bacterium. However, all pregnancies were normal and the puppies were healthy. The cultures of postpartum vaginal discharges were negative. The discrepancy between agglutination and ELISA results has been observed previously and could be attributed to the development of non-agglutinating antibodies [12]. The results from the present study confirmed our previous findings showing that, while 2ME-RSAT was useful as screening test in acute cases, ELISA tests had higher diagnostic sensitivity in both acute and chronic phases of the disease [9]. Three of the female puppies born from infected and treated bitches tested negative after reaching puberty, which indicated that their mothers succeeded in breeding without disseminating the disease. Although B. canis was isolated from only six infected dogs, most of the females that yielded negative blood cultures had been treated with antibiotics before sampling, because enrofloxacin was administered in response to the abortion. The two dogs that became serologically positive in the second sampling had also received antimicrobial prophylaxis before the blood culture sampling. While the results obtained with enrofloxacin in this study were not superior to those obtained with other antimicrobial treatments previously tested, this monotherapy offers the advantage of avoiding the need of combination with streptomycin, which has undesirable toxic effects. An additional advantage of enrofloxacin is the possibility of its use during gestation. Although some textbooks list this drug as not recommended during gestation, this limitation is not mentioned by the manufacturers. Notably, the litters born in this study did not show any health problems. In conclusion, enrofloxacin is an alternative drug to be used in the treatment of canine brucellosis. Its
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efficacy was similar to that described for streptomycin combined with tetracycline or minocycline. Acknowledgements Our thanks to Dr. Nidia Lucero from Instituto Nacional de Microbiologı´a and ‘‘Dr. C.G. Malbra´n’’ for carrying out the bacteriological cultures. References [1] Mateu de Antonio EM, Martin M. In vitro efficacy of several antimicrobial combinations against Brucella canis and Brucella melitensis strains isolated from dogs. Vet Microbiol 1995;45: 1–10. [2] Terakado N, Ueda H, Sugawara H, Isayama Y, Koyama N. Drug susceptibility of Brucella canis isolated from dogs. Jpn J Vet Sci 1978;40:291–5. [3] Castro RF, Carmichael LE. Brucella canis infection in dogs: treatment trials. Rev Latinoam Microbiol 1981;23:75–9. [4] Nicoletti P. Further studies on the use of antibiotics in canine brucellosis. Compend Cont Ed Pract Vet 1991;13(944):946–7.
[5] Zoha SJ, Walsh R. Effect of a two-stage antibiotic treatment regimen on dogs naturally infected with Brucella canis. J Am Vet Med Assoc 1982;180:1474–5. [6] Lewis Jr GE, Crumrine MH, Jennings PB, Fariss BL. Therapeutic value of tetracycline and ampicillin in dogs infected with Brucella canis. J Am Vet Med Assoc 1973;163:239–41. [7] Johnson CA, Bennett M, Jensen RK, Schirmer R. Effect of combined antibiotic therapy on fertility in brood bitches infected with Brucella canis. J Am Vet Med Assoc 1982;180:1330–3. [8] Carmichael LE, Joubert JC. A rapid slide agglutination test for the serodiagnosis of Brucella canis infection that employs a variant (M ) organism as antigen. Cornell Vet 1987;77:3–12. [9] Wanke MM, Delpino MV, Baldi PC. Comparative performance of ELISA assay using cytosolic or outer membrane antigens of Brucella for the serodiagnosis of canine brucellosis. Vet Microbiol 2002;88:367–75. [10] Goldbaum FA, Velikovsky CA, Baldi PC, Mo¨rtl S, Bacher A, Fossati CA. The 18 kDa cytoplasmic protein of Brucella spp., an antigen useful for diagnosis, is a lumazine synthase. J Med Microbiol 1999;48:833–9. [11] Carmichael LE, Shin SJ. Canine brucellosis: a diagnostician’s dilemma. Semin Vet Med Surg (Small Anim) 1996;11:161–5. [12] Parma AE, Santisteban G, Margni RA. Analysis and in vivo assay of B. abortus agglutinating and non-agglutinating antibodies. Vet Microbiol 1984;9:391–8.