1 Brucella canis: Frequency of antibodies present in dogs in a brazilian capital

2 Brucella canis: Frequência de anticorpos presentes em cães em uma capital

3 brasileira

5 Resumo

6 A brucelose é uma doença infecciosa e cosmopolita que afeta animais domésticos,

7 animais selvagens e humanos, por isso é considerada uma zoonose. Portanto, um estudo

8 foi realizado com animais em idade reprodutiva em uma capital brasileira. Um total de

9 591 amostras de soro foram utilizadas de 1 a 7 anos de cães não castrados, sem distinção

10 de sexo ou raça. As amostras foram submetidas a um teste de imunodifusão em gel de

11 ágar usando o antígeno Brucella ovis, a técnica mais utilizada no Brasil nestes casos.

12 Entre os animais selecionados, 44 animais apresentaram positividade para Brucella canis.

13 Concluiu-se que a brucelose está presente na população canina da cidade, mas ainda não

14 veem ser diagnosticados corretamente pelas clínicas veterinárias, o que representa um

15 risco para a saúde pública, uma vez que é uma zoonose.

16 Palavras-chave: Brucelose, caninos, aborto, orquite, zoonoses.

17

18 Abstract

19 Brucellosis is an infectious, cosmopolitan disease that affects domestic animals, wild

20 animals and humans, so it is considered a zoonoses. Therefore, a study was carried out

21 with of animals in reproductive age in a Brazilian capital. A total of 591 serum samples

22 were used from 1 to 7 years of age non-castrate dogs, without distinction of sex or race.

23 The samples were submitted to an agar gel immunodiffusion test using Brucella ovis

24 antigen, the most used technique in Brazil in these cases. Among the selected animals, 44

1
 1 animals showed positivity for Brucella canis. It was concluded that brucellosis is present

2 in the canine population of city, but they still do not see it being properly diagnosed by

3 the Veterinary Clinics, which poses a risk to public health, since it is a zoonoses.

4 Keywords: Brucellosis, canine, abortion, orchitis, zoonoses.

5 Introduction

6 Canine brucellosis is an infectious, systemic disease of a global distribution

7 characterized by presenting a prolonged bacterium of zoonotic character. Dogs affected

8 by Brucella canis infections have several reproductive disorders, with or without obvious

9 clinical signs. In recent decades, social changes and urbanization of the human population

10 have favored an increase in the canine population in developing countries (1). This

11 increase, associated with the sentimental and emotional relations of the man with the dog,

12 has implications in public health, since the animal can be responsible for the transmission

13 of several zoonoses, among them, the brucellosis (2).

14 The disease is caused by bacteria belonging to the genus Brucella, they are

15 obligate intracellular organisms. There are different Brucella species, which exhibit

16 preferences by hosts and differ in severity of disease caused (3).

17 For dogs, there are two serotypes that are the most pathogenic, being Brucella

18 abortus and Brucella canis, causing chronic or subclinical disorders difficult to diagnose

19 by similarity to several other diseases (4). Brucella canis is the etiological agent

20 considered important epidemiologically for establishing inter-relations of the canine

21 population with humans, mainly, the contact between dogs and children in the family

22 environment (4).

23 Serological tests for the detection of antibodies to Brucella are the best means

24 of detecting infection. Among the most widely used serological tests in the diagnosis of

2
 1 Brucella canis in dogs, the most used technique in Brazil is the AGID (agar gel

2 immunodiffusion), in which a lipopolysaccharide antigen extracted from Brucella ovis is

3 used. The sharing of antigens among Brucella canis and Brucella ovis makes it possible

4 to use indistinct reactives produced from these two microorganisms for the diagnosis of

5 brucellosis in canines and sheeps. As this represents a finding of great relevance in public

6 health, since it is a zoonoses, companion animals should be tested, after all, canine

7 brucellosis assumes growing importance in large urban centers (6).

8 In the studied capital, canine brucellosis has not yet been diagnosed

9 and there is no case report of this zoonoses in dogs. Therefore, the objective of this study

10 was to detect if the disease occurs in dogs of this city with clinical signs of the disease or

11 not. This research was carried out using anti-Brucella canis antibodies in serological tests,

12 thus contributing to a better knowledge of this zoonoses in the metropolitan regions.

13 The epidemiological study was carried out in a Brazilian capital to detect a

14 situation in zoonoses in the Metropolitan Region, dogs of the medical clinic of the of

15 Veterinary Teaching Hospital, as well as private clinics in the city, from January 2013 to

16 March 2013.

17 Materials and methods

18 The dogs were randomly selected, without regard to complaint or clinical signs.

19 Were used 591 animals between the age of 1 to 7 years, non-castrate, without distinction

20 of sex or race, with the consent of the person responsible and approval of the Bioethics

21 Committee, in accordance with current rules. Information on age, gender and racial

22 pattern were recorded on an individual file and the clinical examination was conducted

23 by Veterinarians. For serological analysis, blood samples were collected by venipuncture

24 of the jugular vein after antisepsis with 2% iodinated alcohol, with the use of a vacuum

3
 1 system in tubes without anticoagulant which were identified with data concerning the

2 owner and the animal. The tubes were held for 1 to 2 hours to coagulate at room

3 temperature, then stored at 4 ° C overnight and then subjected to centrifugation at 2500

4 rpm for 10 minutes to obtain serum that were stored at -20 ° C.

5 Agar gel immunodiffusion (AGID) was performed with antigen extracted from

6 the Brucella ovis cell wall (Tecpar, Paraná, Brazil) according to a method described in

7 the literature (7), in the Laboratory of Pathophysiology of Female Reproduction on

8 Veterinary Teaching Hospital. The agar gel was prepared using 1.2 g of Noble agar, 5 ml

9 of Borate buffer pH 8.3, and 93 ml of 10% NaCl. Then, 4.5% of the gel was distributed

10 in Petri dishes. After solidification, the gel was drilled with six-hole peripheral rosettes

11 and a central one, with holes 6 mm in diameter and 2.5 mm between the edges. The

12 orifices were then filled with the antigen, positive control serum, serum to be tested and

13 the plates kept in a humid chamber at room temperature.

14 The readings were performed with 24, 48 and 72 hours, using indirect lighting

15 system and dark background. The interpretation was performed by observing the

16 precipitation line formation between the test serum and the control serum positive with

17 the antigen. The test serum whose precipitation lines showed identity with the lines

18 formed by positive control serum was considered positive. The serum considered negative

19 were those in which there was no formation of precipitation lines or the lines formed in

20 the presented the identity with those of the positive control serum.

21 The sample size was calculated by the Epi Info 7.0 program, considering an

22 expected prevalence of 50% (corresponding to a disease of unknown in a defined

23 population) in an estimated population of 89,729 dogs, with a confidence level of 95%

24 and margin error of 5%, which resulted in 384 animals. In the present study, 591 samples

4
 1 of dogs domiciled in the urban area of the city were used. The associations between canine

2 brucellosis is sex, age and race of the animals, were analyzed by the chi-square test is the

3 Fisher's test, with significance level of 0.05. All statistical analyzes of GraphPad Prism

4 software, version 6.0.

5 Results and discussion

6 A total of 591 dogs were tested, and all the records of the animals contained

7 data regarding gender, age and race, and the percentage distribution of the main breeds

8 present in the study population was shown graphically (Figure 1). The sampling used in

9 this study is enough to confirm that the disease is present in the city, since the sample

10 estimate, considering an expected prevalence of 50%, reached a result of 384 samples and

11 in the present were study 591 Samples. Several studies use the same sample calculation

12 (2, 8).

13 Of the 591 samples of canine serum that were tested in the AGID test, 44 were

14 positive, meaning a frequency of infection equal to 7.44% (44/591) in the city. This

15 frequency was higher than other studies in Brazil (8, 9, 10), and outside (11), but are

16 results closer to those of other Brazilian studies (12, 20). Probably, the difference between

17 the frequency found in this work and those cited in other studies is due to the fact that

18 there are differences between the populations studied, region, and sample design. In order

19 to corroborate this hypothesis, it is observed that in a study with the same test used in the

20 present study, and a similar population sample, the result was quite close to that found in

21 this study (7.4%) (12).

22 The test used in this research, AGID was effective as regards the identification of

23 dogs positive for infection. This serological test is the most used in Brazil as an initial test

24 for the detection of positive dogs for Brucella canis (13). Although the test used is

5
 1 considered reliable, there are citations of occurrence of false positives and false negatives

2 (14). Thus, it is suggested that further researches with more sensitive and specific tests,

3 such as PCR, complement fixation and bacteriology (14).

4 The sex, age, and races (Table 1) of the studied canine population (591 dogs)

5 reflect the clientele of the analyzed service where there is balance between the sexes and

6 the dogs without defined breed and adults predominate in the clinical routine (15).

7 Regarding the breeds, other research has shown similar results (13), which agrees with

8 the breeds most attended in Brazilian services (15).

9 Of the 44 reagent samples for Brucella canis infection, 20 (45.45%; 20/44)

10 samples were from male dogs and 24 (54.54%; 24/44) from females (Table 1). It was

11 observed that there was no statistically significant difference between the frequency of

12 reactive animals to the AGID test for canine brucellosis in relation to sex (x2 = 0.44; gl

13 = 17; P.0.5). The absence of a statistically significant difference in sex is the most

14 common in this disease (13, 16). Thus, in the present study there was no significant

15 difference between the sexes, but the absolute number of female was slightly higher. This

16 can be explained by the fact that males usually cover several females, when in the mating

17 period (1). In a Brazilian study, the authors reported that the frequency was higher in

18 males. However, sampling of male dogs was much higher than the number of females,

19 which raises doubts about the reported results (8).

20 In the present study the canine population was grouped in age groups from 1 to

21 5 years and from 5 to 10 years, for better organization and distribution of data, and

22 55.32%% of dogs being between 1 and 5 years old (327/591) and in the age group 5 to

23 10 years old 44.67% (264/591) of the dogs in the sample (Table 2). In the first group (1-

24 5 years, there are 29 positives (29/327; 8.86%), and in the second group (5-10 years) there

6
 1 are 15 positives (15/264; 5.68%). There was no statistically significant difference

2 between the ages tested, which means that dogs of any age are likely to be infected with

3 the bacteria. However, a small difference was observed between the age groups, where

4 the highest percentage of positives is in the range of 1 to 5 years. Similar data have been

5 recorded in other studies (1, 16), are probably related to the fact that in this age group the

6 animals are in their sexual maturity, and generally have contact with more sexual partners,

7 infection among themselves (17).

8 There was no statistically significant difference for the association between the

9 rates and the animals reactive to agar gel immunodiffusion in canine brucellosis (x2 =

10 14.27; gl = 17; P.0.5), in the population sample studied (Table 3). The serological test of

11 agar gel immunodiffusion was efficient and sensitive, being indicated as one of the

12 diagnostic methods for canine brucelosis. The AGID test should be used as a screening

13 test for the diagnosis of canine brucellosis by Brucella canis (2). Knowing that the

14 technique used may result in some false positives and negatives, consideration should be

15 given to this type of interference in the results, it is necessary to carry out more detailed

16 studies, aiming the development, standardization of more specific, sensitive, preferably

17 quick, practical and economically viable technical procedures. Considering that

18 brucellosis is still, the most prevalent zoonoses in the world (18), these measures would

19 be useful in order to implement the early diagnosis, control and treatment of this infection

20 in order to reduce reproductive losses in the kennels and also a possible transmission to

21 humans.

22 Regarding the clinical signs, of the 44 positive animals, it was observed that the

23 most were asymptomatic (56.8%; 25/44). Among the symptomatic dogs (20.4%; 9/44),

24 the majority (89%; 8/9) presented only lymphadenopathy as a clinical sign. One bitch had

7
 1 pyometra. In ten dogs (22.8%; 10/44) it was not possible to recover the clinical signs

2 because the clinical records contained incomplete data. The absence of clinical signs

3 detected in many animals is probably due to the fact that the clinical signs of the disease

4 in dogs are not very evident (14). Lymphadenopathy observed as a single clinical sign in

5 eight animals is common in the disease, but it also appears in other endemic diseases in

6 city as visceral leishmaniasis and erliquiosis (14), complicating the diagnosis of canine

7 brucellosis. Only one animal presented reproductive clinical signs (pyometra). However,

8 most veterinarians do not relate brucellosis with pyometra, not considering Brucella as

9 etiologic factor of pyometra in bitchs (15).

10 Conclusion

11 The results obtained from this research demonstrate that canine brucellosis is

12 present in the canine population of the city and had not yet been detected or diagnosed to

13 date. Most of the dogs tested in this study were asymptomatic for Brucella canis infection.

14 Although the total number of animals evaluated is not expressive enough to represent the

15 canine population of the metropolitan region, the frequency of 7.44% should be taken

16 into account, since it indicates, even if small, a portion of the canine population of city

17 may be playing the role of reservoir for Brucella canis, exposing not only other dogs but

18 also humans to the risk of infection.

19 References

20 1. Souza LA. Prevalence of Brucella canis infection in Belo Horizonte - MG. Rev bras

21 med vet 2002; (3): 13-15.

22 2. Azevedo SS, Vasconcellos AS, Keid LB, Grasso LMPS, Pinheiro RS, Mascolli R,

23 Alves CJ. Comparison of three serological tests applied to the diagnosis of canine

24 infection by Brucella canis. Braz jour vet res anim scien 2004; 41(2) 1-14.

8
 1 3. Hirsh DC, Zee YC. Veterinary Microbiology. 2 st ed. Rio de Janeiro: Guanabara

2 Koogan, 2003.

3 4. Carmichael LE. Canine brucellosis. Isolation, diagnosis, transmission. Proc US

4 Livestock Sanit Ass 1968; 71: 517-527.

5 5. Wallach JC, Giambartolomei GH, Baldi PC, Fossati, CA. Human Infection with M -

6 Strain of Brucella canis. Emer infec dis 2004; 10(1): 146-148.

7 6. Maia GR, Rossi CRS, Abbadia F, Vieira DK, Moraes IA, Prevalence of canine

8 brucellosis in the cities of Rio de Janeiro and Niterói - RJ. Rev bras reprod anim 1999;

9 23(3): 425-427.

10 7. Alton GG, Jones, LM, Angus RD, Verger JM. Techniques for the brucellosis

11 laboratory. Paris, FR: INRA: 1988.

12 8. Alves CJ, Alves FA, Gomes AAB, Azevedo SS, Andrade JSL, Santos FA.

13 Epidemiological Aspects of Brucella canis in Patos, Paraíba, Brazil. Cienc anim 2003;

14 13(1) 45-49.

15 9. Almeida AC, Santorelli A, Bruzadelli RMZ, Oliveira MMNF. Seroepidemiology of

16 canine brucellosis caused by Brucella canis and Brucella abortus in the city of Alfenas,

17 MG. Arq bras med vet zoot 2004; 56(2): 275-276.

18 10. Ferreira T, Mandelbaum MA, Marques APL, Torres HM, Figueiredo MJ, Serra CMB,

19 Aquino M.H.C. Serological survey of Canine Brucellosis through the use of external and

20 internal antigen Brucella canis and Brucella ovis. Rev bras cien vet 2007; 3: 167-168.

21 11. Radojičić S. Bruceloza pasa - epizootiološke karakteristike, dijagnostika, terapija i

22 kontrola bolesti. Vet glas 2006; 60(34): 135-145.

9
 1 12. Marassi CD, Moraes IA, Lilenbaum W. Seroprevalence of Canine Brucellosis in the

2 city of Rio de Janeiro by the agar gel immunodiffusion method. Rev bras cien vet 2003;

3 10(1): 63-64.

4 13. Cavalcante LA, Dasso MG, Oliveira FCS, Viegas SARA, Almeida MGAR,

5 Anunciação AVM, Alcântara AC, Bittencourt DVV, Oliveira EMD. Research on

6 Brucella canis antibodies in dogs from the metropolitan region of Salvador. Rev bras

7 saúde prod anim 2006; 7(2): 176-180.

8 14. Wanke MM. Canine Brucellosis. Anim reprod sci 2004; 82/83: 195-207.

9 15. Evangelista LSM, Lopes RRFB, Alves RPA, Gonçalves LMF, Quessada AM. Renal

10 function in bitches with pyometra before and after ovariosalpingohisterectomy. Acta vet

11 bras 2010; 4(3): 153-161.

12 16. Moraes CCG, Megid J, Souza, LC, Crocci AJ. Prevalence of Canine Brucellosis in

13 the microregion of the Serra de Botucatu, São Paulo, Brazil. Arq inst biol 2002; 69(2): 7-

14 10.

15 17. Acha PN, Szyfres B. Brucelosis. In: Zoonoses and communicable diseases common

16 to man and animals. 3st ed. Washington, USA: OPS/OMS; 2001. pp. 28-56.

17 18. Xavier MN, Costa EA, Paixão TA, Santos RL. The genus Brucella and clinical

18 manifestations of brucellosis. Cienc. rural 2009; 39(7) 2252-2260.

10
 Other breeds 40
Weimaraner 6
Shih Tzu 7
Bulldog 7
Dalmatian 7
Brazilian mastiff 9
Matese 11
Pekingese 12
Labrador 13
Dachshund 15
Yorkshire 16
Cocker spaniel 18
Rottweiler 22
Pinscher 27
Pit bull 32
German Shepherd 37
Poodle 104
Mongrel dogs 208

0 50 100 150 200 250

Figure 1 - Distribution of breeds in dogs submitted to serological tests (immunodiffusion

in agar gel) for Canine Brucellosis (n=591).

11
Table 1 - Frequency of dogs reactive to the immunodiffusion in agar gel for canine
brucellosis, according to sex. (n=591)

% Frequency (+/n)
Sex
Positive Negative
Males 6.73 (20/297)a 93.26 (277/297)a
Females 8.16 (24/294)a 91.83 (270/294)a
+ Numbers of positive animals; “n” Number of samples per variable
a
There was no significant difference (p> 0.05) between sex, according to the test χ2

12
Table 2 - Frequency of dogs reactive to the immunodiffusion in agar gel for canine
brucellosis, according to age (n=591).

% Frequency (+/n)
Age group (years)
Positive Negative
1-5 8.86 (29/327)a 91.13 (298/327)a
5 - 10 5.68 (15/264)a 94.31 (249/264)a
+ Numbers of positive animals; “n” Number of samples per variable
a
There was no significant difference (p> 0.05) between age groups, according to the
test χ2

13
Table 3 - Frequency of dogs reactive to the immunodiffusion in agar gel for canine
brucellosis, according to breed. (n=591).

IDGA1
Total
Breed Reagent animals
Number (%)
Number (%)
Mongrel dogs 20 (9.6)a 208 (35.2)
Poodle 7 (6.7)a 104 (17.6)
German Shepherd 2 (5.4)a 37 (6.3)
a
Pit bull 1 (3.1) 32 (6.4)
Pinscher 1 (3.7)a 27 (4.6)
a
Rottweiler 2 (9.1) 22 (3.7)
Cocker Spaniel 0 (0.0)ab 18 (3.0)
a
Yorkshire 3 (18.8) 16 (2.7)
Dachshund 2 (13.3)a 15 (2.5)
ab
Labrador 0 (0.0) 13 (2.2)
Pekingese 0 (0.0)ab 12 (2.0)
Maltese 0 (0.0)ab 11 (1.9)
ab
Brazilian mastiff 0 (0.0) 9 (1.5)
Bulldog 1 (14.3)a 7 (1.2)
ab
Dalmatian 0 (0.0) 7 (1.2)
Shih Tzu 1 (14.3)a 7 (1.2)
a
Weimaraner 1 (16.7) 6 (1.0)
Other breeds 3 (7.5)a 40 (6.8)
Total 44 (7.4) 591 (100.0)
1
Gel immunodiffusion Agar for Canine Brucellosis.
a
There was no significant difference (p> 0.05) between breed, according to the test χ2.
b
No representative of breed was reagent.

14