Introduction

The Brucellosis is a zoonotic infection worldwide whose incidence and prevalence varies from one country to another. The bacteria belonging to the genus Brucella are characterized by being a gram-negative bacillus, no motility, intracellular optional, represented by six previously recognized species: Brucella melitensis, B. abortus, B. ovis, B. canis, B. suis and B. neotomae, able to infect the macrophages of the host where it multiplies and spreads in a systematic way, causing abortion infectious in animals and febrile disease in humans (Gorvel & Moreno 2002; Rivers et al. 2006). Because of the close interaction between the dogs and humans, brucellosis canine also become very important in terms of public health (George & Carmichael 1974; Vargas 1996; Keid et al. 2006).

Materials and Methods

We analyzed 251 samples of blood serum of dogs from a private veterinary clinic in the city, in the period from January to December 2008, the total samples, 136 were males and 115 females, with different age groups ranging from zero to eight years, belonging to various breeds and crossbreds, all from the 40 neighborhood that make the five administrative districts (Belém, Bengui, Entroncamento, Guamá and Sacramenta), which comprises the city of Belém. The animals were randomly chosen and the samples blood were collected by venipuncture of the cephalic or saphenous vein, stored in sterile tubes without anticoagulant, centrifuged at 2.000g for 10 minutes, resulting in the blood serum, which was stored in Eppendorf tubes type of 1 ml and kept under refrigeration to -2° C until the completion of evidence. The sera were tested with the immunodiffusion in agarose gel (AGID), and used "kits" produced by the Institute of Technology of Paraná (TECPAR). The technique was implemented in accordance with the manufacturer's recommendations, using as the antigen protein and lipopolysaccharide of B. ovis, 198 Reo sample. For confirmation, the same technique was applied in 251 sera treated with 2-mercaptoethanol (AGID-ME), as described by Keid (2001). The whole procedure of the study was conducted at the Laboratory for Research and Diagnosis of Animal Diseases-LIDEA, UFPA-Belém Pará.

Results

Of the 251 samples submitted to the AGID test, 50 (19.9%) were positive and 101 were negative (40.2%), however, the same samples tested in the proof of AGID-ME, only three (1.1%) were confirmed and the remaining 248 (98.9%) was negative. Analyzing the results obtained in the AGID test, the total of 136 females, 27 (19.8%) were positive and 115 males, 23 (23.4%) were positive. 28 (24.5%) were seropositive samples of mixed breed animals and 137 dogs of race, 22 (16%) were positive. Samples of animals that had aged zero to two years, 26 (29.2%) were positive, two to four years of the 38 dogs, seven were positive (18.4%), aged from four to six years, eight samples (22.2%) were positive for a universe of 36 and 88 animals that had six years of age onwards, nine were positive (10.2%). For the spatial distribution in the city, from 57 samples that represented the administrative district of Belém, 17 (29.8%) were positive, in the district of Bengui seven animals were positive (15.9%) of a total of 44; the 49 sera of dogs belonging to the district of Entroncamento, nine (18.3%) formed a line of precipitation of antigen with the antibody in the agar; of 56 samples originating in the district of Guamá, 12 samples (21.4%) were positive and district of Sacramenta, from 45 samples, only seven (15.5%) were seropositive. For results in a confirmatory test (AGID-ME), two females (1.4%) were positive and only one (0.8%) was seropositive of the 115 males; of the 114 mongrel dogs, two were positive (1.7%) and animals with specific breed, one (0.7%) was positive, taking into account the age, one (1.1%) sample was positive in the range that covers animals from zero to two years, also a positive for two to four (2.6%) and one (2.7%) of four to six years, to the administrative district Entroncamento, one (2%) sample was positive and 56 dogs from the district of Guamá, two (3.5%) were positive.

Discussion and Conclusions

The results presented in the study, 1.1% (3/251), was expected in relation to other work done in Brazil, Moraes (2000) detected respectively 0.84% (9/1072) and Azevedo et al. (2003) 2.2% (9/410) of positive animals, both in São Paulo state, where these studies, the authors considered positive, only the dogs to testing reagents with mercaptoethanol (Moraes 2000) and reaction-fixing Completion (Azevedo et al. 2003) as confirmatory, and the result obtained in the test of screening, using only the evidence of AGID 19.9% (50/251), similar values were found by Azevedo (2003), 14.2% (90/635) and Keid (2004) with 33.91% (58/171) positive animals. Evaluating the sex itself may be a risk factor for infection by Brucella, was found no discrepancy between the values of males (1.4%) and females (0.8%) positive for the confirmatory test, because the males and females are also at risk of infection, with no predilection for sex, similar results were found by Hubbert et al. (1980), Germano et al. (1987) and Moraes et al. (2002). In terms of age, were the highest percentages of adult animals (six to eight years) with 2.7%, due to sexual maturity and subsequent coverage, as well as the increased possibility of contact with animals infected with age, they animals show (Johnson & Walker 1992, Carmichael & Greene 1998), already in evidence of AGID, the young animals (zero to two years) presented the highest positive (29.2%); factors such as early infection, in dogs phase of bacteremia and especially the cross-reactions with other gram-negative organisms, can influence these serological results for B. canis (Moraes 2000; Keid 2001), so it is necessary to use other confirmatory evidence of character, such as ME-AGID. The mixed breed animals, presented the highest seroreactivity, 1.7% versus 0.7% of animals with specific breed, Carmichael & Kenney (1968) found that the disease caused by Brucella, came to be diagnosed in dogs of the most different races, not only affecting mongrel dogs. Analyzing the spatial distribution for brucellosis, the administrative districts that had higher numbers of positive animals were Entroncamento (2%), highlighting the district of Guamá with 3.5% (2/56), the fact can be closely linked to the neighborhoods that comprise this district, for presenting large number of free markets, low hygiene and greater presence in the streets and abandoned animals, shelters, which the majority without the minimum conditions for providing structure and apart from that the bulk of resident population is low income, and does not provide the care and management of quality for the animals, which favors the contact with animals already infected. According to Lewis & Anderson (1973), the presence of only a portion of the population positive for B. canis is enough to consider a problem of epidemiological importance, considering the character of zoonotic disease. The results should alert at work to professionals in the area of public health and also for veterinary clinical city that should look to the problem and request more often laboratory tests before the suspicion of the disease, thus allowing the control measures are adopted.

References

1.  Azevedo SS, et al. Inquérito sorológico e fatores de risco para a brucelose por Brucella canis em cães do município de Santana do Parnaíba, Estado de São Paulo. Pesq Vet Bras, v.23, n.4, p.156-160, 2003.

2.  Carmichael LE, Greene CE. 1998. Canine brucellosis, p. 248-257. In: Greene C.E. (ed.) Infectious Diseases of the Dog and Cat. 2nd ed. W.B. Saunders, Philadelphia.

3.  Carmichael LE, Kenney RM. 1968. Canine abortion caused by Brucella canis. J. Am. Vet. Med. Assoc. 152(6):605-616.

4.  George LW, Carmichael LE. A plate agglutination test for the rapid diagnosis of canine brucellosis. Am J Vet Res. 1974 Jul;35(7):905-9.

5.  Germano PML, Vasconcellos SA, Ishizuka MM, Passos EC, Erbolato EB. 1987. Prevalência de infecção por Brucella canis em cães da cidade de Campinas-SP, Brasil. Revta Fac. Med. Vet. Zootec. Univ. S. Paulo 24(1):27-34.

6.  Gorvel JP, Moreno E. 2002. Brucella intracellular life: From invasion to intracellular replication. Vet. Microbiol 90, 281-297.

7.  Hubbert NL, Bech-Nielsen S, Barta O. 1980. Canine brucellosis: comparison of clinical manifestations with serologic test results. J. Am. Vet. Med. Assoc. 177(2):168-171.

8.  Johnson CA, Walker RD. 1992. Clinical signs and diagnosis of Brucella canis infection. Comp. Cont. Educ. Pract. Vet. Small Animal 14(6):763-772.

9.  Keid LB, Soares RM, Megid J, Salgado VR, Vasconcellos SA, Richtzenhain LJ. Avaliação do desempenho das provas de soroaglutinação rápida, soroaglutinação rápida com 2-mercaptoetanol e imunodifusão em gel de ágar para o sorodiagnóstico da brucelose canina em cães naturalmente infectados. In: 19a Reunião Anual do Instituto Biológico, 2006, São Paulo-SP. O Biológico, 2006. v. 68.

10. Keid LB, Soares RM, Morais ZM, Richtzenhain LJ, Vasconcellos AS. Brucella spp. isolation from dogs from commercial breeding kennels in São Paulo state, Brazil. Braz. J. Microbiol. vol.35 no.1-2 São Paulo Jan./June, 2004.

11. Keid LB. 2001. Diagnóstico da brucelose canina por Brucella canis. Correlação entre exames clínicos e laboratoriais: imunodifusão em gel de ágar, imunodifusão em gel de ágar com emprego do 2-mercaptoetanol, cultivo e reação em cadeia pela polimerase. Dissertação de Mestrado em Reprodução Animal, Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, SP. 96p.

12. Lewis GE, Anderson JK. The incidence of B. canis antibodies in sera of military recruits. Am. J. Publ. Health, v.63, p. 204-205, 1973.

13. Moraes CCG, Megid J, Souza LC, Crocci AJ. 2002. Prevalência da brucelose canina na microrregião da serra de Botucatu, São Paulo, Brasil. Arqs Inst. Biológico, São Paulo, 69(2):7-10.

14. Moraes CCG. Prevalência de anticorpos anti-Brucella canis em cães da microrregião da serra de Botucatu, estado de São Paulo. 2000. 89f. Dissertação (Mestrado em vigilância sanitária) - Faculdade de Medicina Veterinária e Zootecnia, Universidade Estadual Paulista.

15. Moreno E, Cloeckaert A, Moriyoni I. 2002. Brucella evaluation and taxonomy. Vet Microbiol 90, 209-227.

16. Rivers R, Andrews E, Gonzalez AS, Donoso G, Oñate A. 2006. Brucella abortus: inmunidad, vacunas y estrategias de prevención basadas en ácidos nucleicos. Arch Med Vet 38, 7-18.

17. Vargas AC, Lazzari A, Dutra V, Poester F. Brucelose canina: Relato de caso. Ciênc. Rural, v. 26, p.305-308, 1996.