versión impresa ISSN 1517-8382
Braz. J. Microbiol. vol.42 no.4 São Paulo oct./dic. 2011
Annielle Regina Fonseca FernandesI; Sérgio Santos de AzevedoI,*; Rosa Maria PiattiII; Eliana Scarcelli PinheiroII; Margareth Élide GenovezII; Adílio Santos de AzevedoI; Carolina de Sousa Américo BatistaIII; Clebert José AlvesI
ILaboratório de Doenças Transmissíveis, Unidade Acadêmica de Medicina Veterinária, Centro de Saúde e Tecnologia Rural, Universidade Federal de Campina Grande, Patos, PB, Brasil
IILaboratório de Doenças Bacterianas da Reprodução, Centro de Pesquisa e Desenvolvimento em Sanidade Animal, Instituto Biológico de São Paulo, SP, Brasil
IIIDepartamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, SP, Brasil
To determine the frequency of anti-Brucella canis antibodies in dogs attended in veterinary clinics from Patos, Paraíba State, Brazil, as well as to identify risk factors and to isolate and identify the agent, 193 dogs were used. Agar gel immunodiffusion test (AGID) was used to detect B. canis antibodies in sera. Isolation of B. canis was carried out in blood and bone marrow from seropositive animals. Six animals tested seropositive in AGID, resulting in a frequency of 3.11%. B. canis was isolated from bone marrow of one seropositive animal, with confirmation by PCR. Lack of cleaning of the dog's environment was identified as risk factor (odds ratio = 7.91). This is the first report of isolation of B. canis in dogs from the Northeast region of Brazil.
Key words: Bacterial diseases, canine brucellosis, prevalence, risk factors, microbiological culture
Canine Brucellosis is an infectious disease of zoonotic potential whose etiologic agent is B. canis, a bacterium responsible for impairment of reproductive tract, primarily abortion and sterility in females and orchitis and epididymitis in males (1). Zoonotic potential of the disease should be taking into account due to complex and close relationship with human population, especially children.
Although clinical signs are usually related to disorders of the reproductive tract, in most cases, even it being a disease of systemic character, the animals are apparently healthy (asymptomatic), behaving as important sources of infection (1).
Etiological diagnosis is performed by detection of antibodies in blood serum or isolation of the agent from infected animals as well as molecular methods (6, 12). Bacterial isolation can be done by cultivation of blood and secretions of infected animals and although it provides a definitive diagnosis, this procedure is laborious and time-consuming and may produce false-negative results. Serological tests are more practical, quick and easy to implement enabling the execution of a considerable number of samples. However most serological tests are not specific to B. canis and can give false-positive results. Molecular methods such as polymerase chain reaction (PCR) have been widely used for the diagnosis of canine brucellosis enabling the detection of DNA of bacteria in several samples (8).
The aim of this work was to determine the frequency of anti-B. canis antibodies in dogs attended in veterinary clinics from Patos, Paraíba State, in the Northeast region of Brazil, as well as to identify risk factors and to confirm the infection by microbiological culture and PCR.
Dogs ≥ 3 months-old (n = 193) assisted in the Veterinary Medical Center Dr. Leonardo Torres and in the Clinic of Small Animals of the Veterinary Hospital of the Federal University of Campina Grande, Patos, Paraíba, Brazil, from July 2008 to April 2009, were used and selected with the consent of the owners. To identify risk factors, epizootiological questionnaires with closed questions were supplied to each dog owner. These questionnaires were administered by the same interviewer.
Blood (4 mL) was collected without anticoagulant from each dog. Blood samples were centrifuged (2000 X g for 15 min) and sera were stored at -20 ºC prior to testing. From seropositive animals, blood and bone marrow samples were collected. Blood (4 mL) was collected from each animal by jugular venal puncture with sodium citrate as anticoagulant. An aliquot (2 mL) was submitted to bacterial isolation. Bone marrow was collected by iliac crest puncture and 2 mL were used for bacterial isolation.
Sera were tested by AGID test using Brucella ovis surface antigen, produced in the Instituto Tecnológico do Paraná (Tecpar, Paraná, Brazil). The tests were performed according to the laboratory recommendations, except for the substitution of agarose by 1% agar Noble (Difco, Detroit, MI, USA).
Risk factors analysis was performed in two steps: univariate and multivariate analysis. Univariate analysis was performed using the Chi-square test or Fisher's exact test (13), and those variables that presented p ≤ 0.20 were used for multiple logistic regression. The multivariate analysis was then performed, using the stepwise forward method (5). The significance level in multivariate analysis was 5%. The tests were performed using the SPSS for Windows software package, version 13.0.
For blood and bone marrow cultures 2 mL of blood with sodium citrate and 2 mL of bone marrow were inoculated in Castañeda medium and incubated at aerobic atmosphere (37ºC for 30 days) (1). After growth, colonies were cultured on Brucella agar plates and incubated at aerobic atmosphere at 37ºC for five days for bacterial identification. Genus characterization was performed using Gram staining and identification of the biochemical profile: catalase, oxidase, citrate, nitrate reduction, motility, H2S production and urease (7).
DNA extraction of isolated bacteria was performed by boiling method (10, 11). A 2.3 x 109 bacteria/mL suspended in 1000 µL of sterile bi-distilled water, corresponding to 8 on the MacFarland scale, was heated for 10 min to 99 ºC, and further used for PCR assay. DNA obtained was stored at -20 ºC till amplification.
For the reaction primers B4 (5'-TGGCTCGGTTGCCAATATCAA-3') and B5 (3'-CGCGCTTGCCTTTCAGGTCTG-5') were used to amplify a 223 bp sequence of the gene encoding the periplasmic immunogenic protein BCSP31 specific for Brucella spp. The amplification reaction mixture was prepared in a volume of 50 µL containing 200 μM of each deoxynucleoside tryphosphate, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 0.5 mM of each primer, 1.5 U platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) and 5 µL of template DNA. The reaction was performed in a DNA thermal cycler (MJ Research PTC 200 DNA engine, Watertown, MA, USA) without mineral oil. Ultrapure water was used as negative control and Brucella abortus strain ATCC 544 as positive control. After an initial denaturation at 95ºC for 2 min, the PCR profile was set as follows: 30s of template denaturation at 95ºC, 30s of primer annealing at 62ºC and 30s of primer extension at 72ºC, for a total of 40 cycles, with a final extension at 72ºC for 5 min. The samples were analyzed by electrophoresis in a 2% agarose gel and then stained with ethidium bromide (0.5 mg/mL). The DNA bands were visualized under UV light.
Six animals tested positive at AGID test, resulting in a frequency of 3.11%. In the univariate analysis to determine risk factors for B. canis infection, variables cleaning of the dog's environment and walking with the dogs were selected (Table 1). Risk factor identified in multivariate analysis by logistic regression was lack of cleaning of the dog's environment (odds ratio = 7.91, 95% CI = 1.50 - 41.72, p = 0.015), which is plausible from the biological standpoint, since the routine cleaning of the environment with common disinfectants may reduce the survival of bacteria eliminated by infected dogs in the environment, especially in case of parturition or abortion. Therefore, it is suggested that this practice must be commonly adopted in order to avoid exposure of other animals and humans to the risk of infection.
Bacteria with morphostaining and biochemical characteristics similar to Brucella spp. was isolated from bone marrow of one seropositive animal. Brucella DNA was extracted from isolated colonies using the boiling procedure and the extracted DNA was amplified using genus-specific primers for Brucella spp.
In this study, the agent was isolated from the bone marrow of one seropositive animal. Although blood is considered the material of choice for the isolation due to long period of bacteremia, B. canis can also locate in other lymphoid organs, so that it can be recovered by bone marrow aspirates in the absence of positive blood cultures (6).
The biochemical profile of the isolate was: catalase positive, oxidase positive, citrate negative, nitrate reduction positive, motility negative, H2S production negative and urease positive. The results of biochemical tests are consistent with those obtained in other studies (3, 4, 7) with the exception of urease positive. Several studies of isolation of B. canis found some variations when biochemical tests were performed as Flores Castro et al. (2), which had samples that did not reduce nitrate. Larsson and Costa (9) examined 27 dogs, and 3 had positive blood cultures. When biochemical tests were performed the strains showed production of H2S positive.
The findings described above indicate the presence of B. canis infection among pet dogs from Patos, Paraíba State, Brazil, and this work is the first to report the isolation of B. canis in dogs in the Northeast region of Brazil, with confirmation by PCR. Since human infection with B. canis has been reported (12), attention should be paid to possible human infection with this zoonosis through pet dogs and preventive measures must be taken to prevent the transmission. It is suggested that routine cleaning of the dog's environment should be adopted in order to avoid exposure of other animals at risk of infection.
1. Carmichael, L. E.; Greene, C. E. (1998). Canine brucellosis. In: Greene, C.E. (ed). Infectious diseases of the dog and cat. 2. ed. W.B. Saunders, Philadelphia, p.248 257. [ Links ]
2. Flores-Castro, R.; Suarez, F.; Ramirez-Pfeiffer, C.; Carmichael, L. E. (1977). Canine Brucellosis: Bacteriological and Serological Investigation of Naturally Infected Dogs in Mexico City. J. Clin. Microbiol. 6(6): 591-597. [ Links ]
3. Godoy, A. M.; Peres, J. N.; Barg, L. (1977). Isolamento de Brucella canis em Minas Gerais, Brasil. Arq. Esc. Vet. Univ. Fed. Minas Gerais 29(1): 35-42. [ Links ]
4. Gomes, M. J. P.; Driemeier, D.; Soares, H. C.; Bastos, C. D.; Canto, S. P.; Brum, M.; Rossi, A. C.; Corbellini, L. G. (1999). Brucella canis: isolamento em um cão com epididimite e orquite - relato de caso. Clínica Veterinária 4(18): 17-20. [ Links ]
5. Hosmer, D.W.; Lemeshow, S. (2000). Applied logistic regression. John Wiley & Sons, New York, 375p. [ Links ]
6. Johnson, C.A.; Walker, R.D. (1992). Clinical signs and diagnosis of Brucella canis infection. Comp. Contin. Educ. Pract. Vet. 14, 763-772. [ Links ]
7. Keid, L.B.; Soares, R.M.; Morais, Z.M.; Richtzenhain, L.J.; Vasconcellos, S.A. (2004). Brucella spp. isolation from dogs from commercial breeding kennels in São Paulo state, Brazil. Braz. J. Microbiol. 35(1-2): 161-166. [ Links ]
8. Keid, L.B.; Soares, R.M.; Vieira, N.R.; Megid, J.; Salgado, V.R.; Vasconcellos, S.A.; Costa, M.; Richtzenhain, L.J. (2007). Diagnosis of canine brucellosis: comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer. Vet. Res. Commun. 31, 951-965. [ Links ]
9. Larsson, M. H. M. A.; Costa, E. O. (1980). Isolation of Brucella canis. Int. J. Zoonoses 7(2): 125-130. [ Links ]
10. Nishimura, M.; Nukina, M.; Yuan, J.M.; Shen, B.Q.; Jian, J.M.; Ohta, M.; Saida, T.; Uchiyama, T. (1996). PCR-based restriction fragment lengt polymorphism (RFLP) analysis and serotyping of Campylobacter jejuni isolates from diarrheic patients in China and Japan. Fems Microbiol. Let. 142, 133-138. [ Links ]
11. On, S.; Harrington, C.S. (2001). Evaluation of numerical analysis of pfge-dna profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16S rDNA sequencing methods. J. Appl. Microbiol. 90, 285-293. [ Links ]
12. Wanke, M.M. (2004). Canine brucellosis. Anim. Reprod. Sci. 83, 195-207. [ Links ]
13. Zar, J.H. (1999). Biostatistical analysis. 4. ed. Prentice Hall, Upper Saddle River, 663p. [ Links ]
Submitted: April 24, 2010; Approved: June 06, 2011.
* Corresponding Author. Mailing address: Unidade Acadêmica de Medicina Veterinária, Centro de Saúde e Tecnologia Rural, Universidade Federal de Campina Grande, Patos, Paraíba, Brazil.; E-mail: email@example.com