Serodiagnosis of brucellosis in mules from Southwestern Goiás State, Brazil. Sorodiagnóstico da brucelose em muares do Sudoeste Goiano, Brasil

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1 DOI: / v38n6p3669 Serodiagnosis of brucellosis in mules from Southwestern Goiás State, Brazil Sorodiagnóstico da brucelose em muares do Sudoeste Goiano, Brasil Rodrigo Garcia Motta 1* ; Lorrayne de Souza Araújo Martins 2 ; Priscila Alonso dos Santos 3 ; Igor Garcia Motta 4 ; Maria Cristina de Oliveira 5 ; Márcio Garcia Ribeiro 6 Abstract Equine brucellosis is characterised as a zoonotic, chronic infectious disease caused by gram-negative bacteria of the genus Brucella. The economic losses from this infection are considered lower in equine species than those assessed in other livestock species because the occurrence of equine reproductive disorders is rare compared to those in cattle, buffaloes, pigs, and small ruminants. In equines, this disease manifests mainly as chronic joint damage. Lesions suggestive of the disease in equines are represented by ligament inflammation, particularly bursitis, in he scapulae, although asymptomatic cases may occur as well. However, data about the prevalence of brucellosis in mules is poorly understood. In this scenario, the present study investigated the serodiagnosis of brucellosis in mules from the southwestern region of the state of Goiás, Brazil, where equine breeding is common. In total, 626 serum samples were collected from 404 (64.54%) female mules, 208 (33.22%) male mules, and 14 (2.24%) male hinnies. The Rose Bengal test (RBT) used for the initial screening identified 4.15% (26/626) positive animals. When these 26 samples were subjected to the 2-mercaptoethanol (2-ME) test to confirm the diagnosis, 3.15% (22/626) positive samples were detected, showing a good concordance (84.6%) between the RBT and 2-ME test. All 14 females and 8 male positive mules in the 2-ME test were asymptomatic. Among these 22 2-ME-positive animals, 86.36% ranged from 6 to 15 years of age, with significant differences (p=0.036) in prevalence among older animals. Furthermore, 77.27% of the 2-ME-positive animals were kept in pastures with cattle. The high prevalence of brucellosis among older mules reinforces the chronic behaviour of diseases in livestock. In addition, the high frequency of disease in mules with a history of contact with cattle indicates that the pathogen was probably transmitted from cattle-to-mules by contact with contaminated pastures or water after abortion of infected cattle. To our knowledge, this is the first study of the comparative serodiagnosis of brucellosis in mules in Brazil with a large number of animals using both the RBT and 2-ME test. Key words: Equines. Brucellosis. Antibodies. Epidemiology. Prevalence. 1 Pós-Doutorando, Universidade Estadual Paulista Júlio de Mesquita Filho, FMVZ, UNESP, Botucatu, SP, Prof. Dr., Faculdade de Medicina Veterinária, Universidade de Rio Verde, UniRV, Rio Verde, GO, Brasil. rgmottafmvz@gmail.com 2 Discente, Curso de Mestrado, Programa de Pós-Graduação em Tecnologia de Alimentos, Instituto Federal de Educação Ciência e Tecnologia Goiano, IFGoiano, Rio Verde, GO, Brasil. lorraynevet@hotmail.com 3 Profª Drª, Curso de Engenharia de Alimentos, IFGoiano, Rio Verde, GO, Brasil. prialonso@yahoo.com.br 4 Discente, Curso de Mestrado, Programa de Pós-Graduação em Reprodução Animal, Universidade de São Paulo, USP, Pirassununga, SP, Brasil. igorgmotta@hotmail.com 5 Profª Drª, Curso de Medicina Veterinária, Universidade de Rio Verde, UniRV, Rio Verde, GO, Brasil. mcorv@yahoo. com.br 6 Prof. Livre, Departamento de Higiene Veterinária e Saúde Pública, FMVZ/UNESP, Botucatu, SP, Brasil. mgribeiro@ fmvz.unesp.br * Author for correspondence Received: May 26, Approved: Sept. 29,

2 Motta, R. G. et al. Resumo A brucelose dos equídeos caracteriza-se como doença infecto-contagiosa crônica, com caráter zoonótico, causada por bactéria gram-negativa do gênero Brucella. Na espécie equina, os prejuízos econômicos advindos desta infecção são considerados menores, quando comparados a outras espécies de interesse zootécnico, visto que não são reportados transtornos da esfera reprodutiva como descrito em bovinos, bubalinos, suínos e pequenos ruminantes. Nos equídeos, esta enfermidade manifesta-se sob a forma de lesões articulares crônicas. As lesões sugestivas da doença em cavalos são representadas por inflamações em ligamentos, particularmente bursite inter-escapular, embora também sejam identificados portadores assintomáticos. A soroprevalência da brucelose em muares é pouco estudada. Neste cenário, foi investigada a prevalência de imunoglobulinas anti Brucella abortus em muares provenientes da região sudoeste de estado de Goiás, Brasil, onde é comum a criação destes animais. Foram coletadas 626 amostras de soro obtidas de 404 (64,54%) mulas, 208 (33,22%) burros e 14 (2,24%) bardotos. O teste do Antígeno Tamponado Acidificado (ATA) utilizado na triagem resultou em 26 (4,15%) animais positivos que, quando submetidos a prova confirmatória de 2-ME identificou 22 (3,51%) positivos. Destes, 14 eram mulas e 8 burros, todos assintomáticos. Ainda, 86,36% dos animais positivos no 2-ME situavam-se entre 6 e 15 anos de idade, com diferença estatisticamente significativa (p=0,036) para a doença em animais mais velhos. Destaca-se que 77,27% dos animais positivos ao teste do 2-ME eram criados em consórcio com bovinos. O perfil epidemiológico da infecção em muares adultos reforça o comportamento crônico da brucelose. Ainda, foi possível comprovar que os animais positivos, apresentavam histórico de contato próximo com bovinos, indicando que os equídeos provavelmente foram infectados pelo contato com água e pastos contaminados após o abortamento de vacas infectadas. Dentre a literatura consultada descreve-se pela primeira vez no Brasil estudo sorológico da brucelose em grande número de mulas, utilizando os testes do ATA e 2-ME. Palavras-chave: Equídeos. Brucelose. Anticorpos. Epidemiologia. Prevalência. Introduction Equines have coexisted with humans for millennia and are used for transportation, leisure, food, entertainment, sport, and in equine-assisted therapy for children, adults, and elderly people with special needs (RIBEIRO et al., 2008). Brazil ranks fourth in the number of equines, with approximately ten million animals, surpassed only by China, Canada and Mexico, according to the Brazilian Confederation of Agriculture and Livestock Instituto Brasileiro de Geografia e Estatística (IBGE). These animals are mostly found in the Northeast (24.8%), Southeast (24.6%) and Central West (20.4%) regions. The state of Goiás accounts for 7.8% of equids in the Central West region of Brazil, and Southwestern Goiás has a population of approximately 37,900 head (IBGE, 2016). Brucellosis is caused by well-recognised gramnegative bacteria of the genus Brucella and is characterised as a zoonotic, chronic infectious disease in domestic animals (NARDI JÚNIOR et al., 2015; MEGID et al., 2016). In equines, the economic losses caused by species of Brucella are considered lower than those observed in other livestock because reproductive disorders in equines are a rare clinical condition (LANGONI; SILVA, 1997; OMER et al., 2000; RIBEIRO et al., 2008). Brucella species may be divided into two antigenically different groups: smooth (B. abortus, B. melitensis and B. suis) and rough (B. ovis and B. canis) (QUINN et al., 2011; MEGID et al., 2016). Organisms of the Brucella genus lack specificity regarding their host, although some preferences of animal species exist (OMER et al., 2000; NARDI JÚNIOR et al., 2015). B. abortus preferentially affects cattle and equines. B. suis infect predominantly pigs, whereas B. melitensis commonly is causal agent in goats (exotic in Brazil). B. ovis preferentially affects sheep, while B. canis usually infect dogs. Of these, B. melitensis, B. suis, B. abortus and B. canis have recognised zoonotic potential (ACHA; SZYFRES, 2003). 3670

3 Serodiagnosis of brucellosis in mules from Southwestern Goiás State, Brazil B. abortus and occasionally B. suis are the main causative agents of equine brucellosis. In this domestic species, the disease causes chronic joint damage, particularly abscesses of the withers, andrarely abortions. The lesions most suggestive of the equine disease are ligament inflammation and/or bursitis, although asymptomatic cases are reported as well (VIANA et al., 1981; SILVA et al., 2006; RIBEIRO et al., 2008; NARDI JÚNIOR et al., 2015). Equine brucellosis requires euthanasia of infected animals due to its debilitating injuries and also because it is a potential source of infection for other domestic species and humans (RIBEIRO et al., 2003; RADOSTITS et al., 2007; NARDI JÚNIOR et al., 2015). Although the mechanism of transmission of brucellosis among equines is poorly understood, studies have shown that infection is favoured by cohabitation with infected cattle and pigs (ACHA; SZYFRES, 2003; RIBEIRO et al., 2008; KARTHIK et al., 2016); thereby suggesting that transmission occurs through intake of water and food contaminated with vaginal discharge, abortion or placental remains from infected animals (POESTER et al., 2002; NARDI JÚNIOR et al., 2015; RIBEIRO et al., 2008; MEGID et al., 2016). The difficulty in isolating the agent from specific lesions, as well as contamination with opportunistic microorganisms are limiting factors for the microbiological diagnosis of this disease (RIBEIRO et al., 2003; RADOSTITS et al., 2007; NARDI JÚNIOR et al., 2015; MEGID et al., 2016). Considering these limitations, the serological diagnosis of brucellosis has been widely recommended in livestock species, including equines (RADOSTITS et al., 2007). In Brazil, data about the prevalence or diagnosis of brucellosis in equines is commonly recovered from horses (RIBEIRO et al., 2003; ALMEIDA et al., 2007; ARRUDA et al., 2012; DORNELES et al., 2013; OLIVEIRA FILHO et al., 2013) (Table 1). Conversely, serodiagnosis of brucellosis in mules is poorly understood in this country. Therefore, the aim of the present study was to investigate the serodiagnosis of brucellosis among asymptomatic mules in the southwestern region of the state of Goiás, Brazil. Table 1. Prevalence of brucellosis in serum samples from mules of the southwestern region of the state of Goiás, Brazil, 2016*. Animals Number (%) RBT-positive animals (%) 2-ME-positive animals (%) Female Mules 404 (64.54%) A 16 (2.55%) A 14 (2.23%) A Male Mules 208 (33.22%) B 10 (1.59%) A 8 (1.28%) B Male Hinnies 14 (2.24%) C - - Total 626 (100%) 26 (4.15%) 22 (3.51%) *Different lowercase letters in the same rows or columns differ from each other according to the χ2 test (P<0.05). RBT=Rose Bengal test, 2-ME = 2-mercaptoethanol test Materials and Methods Animals and serum samples The study was conducted from April 2015 to December 2016 using 626 mules from different farms of the following counties: Rio Verde, Iporá, Caiapônia, Maurilândia, Castelândia, and Santa Helena, located in the southwestern region of Goiás, Brazil, where equine breeding is common. The samples were collected from asymptomatic animals at the Veterinary Hospital of the University of Rio Verde, Goiás, Brazil, that were included in the University Extension Project Cool Cart-Drivers Don t Abuse Animals. In total, 626 samples containing 10 ml of blood were collected by aseptic venepuncture of the external jugular with a vacuum tube and a siliconised 3671

4 Motta, R. G. et al. container and kept at room temperature until clot retraction. Serum aliquots were transferred to 1.0- ml polypropylene (microcentrifuge) tubes and stored at 20 C (common freezer) until the carried out screening Rose Bengal plate test (RBT) was performed. Only positive samples in the RBT were subjected to confirmation using the 2-mercaptoethanol (2-ME) test. The RBT and 2-ME test were performed according to guidelines of Office International De Epizooties-OIE (OIE, 2012). During the clinical examination, epidemiological data of the owners were investigated to establish the profile of the infected animals. These data were subjected to Pearson parametric correlation analysis and Chi-square tests (significance level of <0.05) to assess the following variables: age, gender, presence of other domestic animals, and positivity in the RBT and 2-ME test (TRIOLA, 2013). In this study, 626 asymptomatic mules were sampled, with ages ranging from 1 to 18 years, including 404 female mules (64.54%), 208 male mules (33.22%) and 14 male hinnies (2.24%). This project was approved by the UniRV Ethics Committee on Animal Use of the University of Rio Verde, Goiás, Brazil (protocol number ). Results and Discussion The RBT identified 4.15% (26/626) of equids as positive, of which 22 animals were also positive using the 2-ME test (22/626=3.51%) (Table 1), resulting in a concordance of 84.6%. The prevalence of brucellosis in equines has been the focus of several studies and ranges from 2 to 15% in the Americas (ACHA; SZYFRES, 2003; STARK et al., 2008). Particularly in Brazil, studies on the serodiagnosis of equine brucellosis have been conducted from 1970 to 2015, with the percentage of positive animals ranging from 1 to 34%, depending on the regions in which the animals were sampled (VIANA et al., 1981; RIBEIRO et al., 2003; CARRAZZA et al., 2010; ARRUDA et al., 2012; CHAVES et al., 2015). Nevertheless, to date, no serological or epidemiological surveys have been conducted exclusively among mules in Brazil. In this context, the studied region in the state of Goiás has a particularly high number of owners who breed mules. Among mules sampled, 77.27% (17/22) of 2-MEpositive animals were kept in pastures with cattle. However, no clinical signs suggestive of brucellosis were observed in any mules, including among seropositive animals. This circumstantial evidence of absence of clinical manifestations of disease could be explained by the hybrid origin of mules, which may result in relative resistance to clinical manifestations of brucellosis, including infections in the reproductive tract or articulations (SILVA et al., 2006). Conversely, in a recent study conducted in Nigeria, vaginal secretions from 6 mares with a history of subfertility described microbiological isolation of B. abortus in 2 animals, representing a rare report of the isolation of pathogen in clinical cases associated with reproductive disorders in equines (BERTU et al., 2015). Nine equines with bursitis of the withers that were seropositive for brucellosis were significantly associated with sharing pastures simultaneously used by cattle, which indicates that co-habitation between domestic species is a risk factor for equine brucellosis (COHEN et al., 1992). The present study showed a significant association (p=0.028) between RBT and 2-ME-positive animals (84.6%). This result is expected because both tests preferentially detect the IgG class of immunoglobulins (Igs), although the 2-ME test is more specific (OIE, 2012). Our findings showed that 86.35% of 2-ME positive mules ranged from 6 to 15 years in age, and significant differences (p=0.042) were found in the diagnosis of brucellosis in older than younger animals. Carrazza et al. (2010) found that the diagnosis of equine brucellosis was more common among animals 3672

5 Serodiagnosis of brucellosis in mules from Southwestern Goiás State, Brazil aged from 10 to 12 years. Likewise, in our mules the highest prevalence of 2-ME-positive (27.30%) was found among animals with an average age of 13.5 years (Table 2). In fact, the older animals have a higher risk of exposure to bacteria from placentas, lochia, foetuses, and placental discharges, which may to contaminate the water and food, representing an important transmission route of pathogen to horses, mules, and donkeys (MEGID et al., 2016). Table 2. Serodiagnosis of brucellosis using the RBT and 2-ME tests in mules, according to age *. Age/years Absolute number (2-ME) Relative number of 2-ME-positive animals by age group Absolute number (RBT) Relative number of RBT-positive animals by age group % A % A % A % A % B % B % B % B % C % C % B % C Total % % *Different lowercase letters in the same rows or columns differ from each other according to the χ2 test (P<0.05). RBT=Rose Bengal test, 2-ME = confirmation or 2-mercaptoethanol test, %=percentage. The dynamics of Igs must be addressed to best understand the method adopted for the diagnosis of brucellosis in mules in this study. The production of Igs in livestock after infection by pathogenic Brucella species is characterized by the occurrence of 4 isotypes: IgM, IgG1, IgG2, and IgA (NIELSEN et al., 2004). Igs are mostly directed against membrane lipopolysaccharides (LPS) of Brucella spp., in the polysaccharide fraction (NIELSEN et al., 2004; RIBEIRO et al., 2008; NARDI JÚNIOR et al., 2015). The IgM response in infected animals occurs a few days after exposure, increasing rapidly, reaching peak value at approximately the second week, before subsequently declining and remaining at detectable levels (NIELSEN et al., 2004). The IgG1 isotype follows a similar pattern, peaking later than IgM (4-6 weeks from infection), usually remaining detectable for long periods (NIELSEN et al., 2004). The IgG class is the most important from the diagnostic standpoint, given its binding specificity, with unique epitopes for the genus Brucella (MEGID et al., 2016). The isotopes IgG2 and IgA appear approximately 2 weeks post-infection and gradually increase but remain at low levels. The IgM class shows agglutination in serological tests (MEGID et al., 2016). This class is predominant in the humoural response in acute infections by Brucella species and after vaccination with the B. abortus B-19 strain. However, this class shows undesirable cross-reactions with other gramnegative bacteria that contain a wall structure similar to that of the genus Brucella (NIELSEN et al., 2004), including Yersinia enterocolitica, Escherichia coli, Salmonella spp., and Pseudomonas aeruginosa (GONZÁLES et al., 2006). Hence, there is a need to use confirmatory serological tests, as proposed in our study with mules. In the RBT test, the B. abortus antigen is acidified (ph 3.65), which limits the agglutination with IgM, prioritising reactions with the IgG class, considered the specific Ig to the disease (NIELSEN et al., 2004). The RBT is considered a sensitivity method and commonly is used as routine (screening) test, although in RBT-positive animals confirmation tests 3673

6 Motta, R. G. et al. are recommended (MEGID et al., 2016). Conversely RBT test, the 2-ME test has higher specificity for the diagnosis of brucellosis (NIELSEN et al., 2004). According to the Brazilian Program of Control and Eradication of Brucellosis and Tuberculosis, positive cattle and buffaloes to RBT and/or 2-ME should be euthanised. Nevertheless, despite the recommendation of euthanasia for equines seropositive for brucellosis, no consensus for the serological diagnosis has been adopted to equids (RIBEIRO et al., 2008). Particularly in Brazil, the serodiagnosis of equine brucellosis has been performed throughout different states (Table 3). A study carried out in the state of Goiás with 52 equines with bursitis of the nape and withers described a high prevalence of positive animals (38/52=73.1%), as opposed mostly Brazilian studies that show a low prevalence of equine brucellosis. In this context, Dorneles et al. (2013) reported 1.76% positive-animals in the 2-ME test for working equines from Mossoró, RN. Furquim et al. (2012) investigated 107 horses and mules serum samples intended for slaughter and detected 4.67% (5/107) positive-animals in the 2-ME test. Similarly, Junqueira Nardi Júnior et al., (2015) founded 1.37% seroprevalence of brucellosis in equines used in cattle farming in Minas Gerais, using the 2-ME test for confirmation. In another study conducted in the Southeast region of Brazil, 123 serum samples were collected from asymptomatic working equines and subjected to the RBT, which identified 6.5% (8/123) positive-animals (ANTUNES et al., 2013). In Pará, a serological study of brucellosis performed with various domestic species showed a 17.85% of prevalence in equines. The authors highlighted a prevalence of 19.14% in cattle, which is very similar to founded in equines, indicating the likely contribution of cohabitation to the interspecies transmission of the pathogen (LOPES et al., 1999). Conversely, Almeida et al. (2007) tested 875 serum samples from equines slaughtered in the South and Southwest regions and observed 2% (15/875) positive-animals using the RBT. In contrast, absence or low levels of anti-brucella spp. antibodies have been described in mules. No positive animals were detected among the 123 draft animals from Paraíba (ANTUNES et al., 2013). Chaves et al. (2015) investigated 415 equines from the state of Maranhão and detected only 0.96% (451) positive-animals to the 2-ME test. Santos et al. (2012) tested 187 working equines of rural areas from Uberlândia county, Minas Gerais, and all animals were serologically negative for brucellosis. Table 3. Serodiagnosis of equine brucellosis in different Brazilian states *. Author Year Location Prevalence (%) Test Species Oliveira et al Rio Grande do Sul 5.3 SAT Horses Jardim et al Goiás 1.4 FAT Horses Viana et al Minas Gerais 0.4 FAT Horses Feitosa et al São Paulo 16.8 SAT Horses Langoni and Silva 1997 São Paulo 0.8 SAT Horses, donkeys Lopes et al Pará 17.8 RBT Horses Silva et al Goiás 71.3 RBT Horses with symptoms suggesting brucellosis (fistulous withers or nape) Almeida et al South and Southwest regions 2.0 RBT Horses Aguiar et al Rondônia ME Horses, mules Stark et al Rio Grande do Sul - RBT Horses continue 3674

7 Serodiagnosis of brucellosis in mules from Southwestern Goiás State, Brazil continuation Author Year Location Prevalence (%) Test Species Araújo et al Minas Gerais 8.1 RBT Horses Carrazza et al Minas Gerais 2.1 RBT Horses Almeida and Silva 2010 Minas Gerais ME Horses Arruda et al Paraíba 0.6 CF Horses Furquim et al Paraná ME Horses (intended for slaughter) Santos et al Minas Gerais - RBT Horses Antunes et al Paraná 6.5 RBT Horses Oliveira Filho et al Paraíba - RBT Horses Dorneles et al Rio Grande do Norte ME Horses Pinho et al Pará ME Horses Chaves et al Maranhão ME Horses, donkeys Junqueira Júnior 2015 Minas Gerais ME Horses, mules Santos et al South region ME Horses, mules *Adapted from Oliveira Filho (2013) (RBT = Rose Bengal plate agglutination test, 2-ME = 2-mercaptoethanol test, FAT = fast agglutination test, SAT = slow agglutination test, CF = complement fixation). Conclusions The high prevalence of brucellosis among older mules reinforces the chronic behaviour of diseases in livestock. In addition, the high frequency of disease in mules with a history of contact with cattle indicates that the pathogen probably is transmitted from cattle-to-mules by contact with contaminated pasture or water after abortion of infected cattle. To our knowledge, this is the first study of the comparative serodiagnosis of brucellosis in mules in Brazil with a large number of animals using both the RBT and 2-ME test. Noteworthy that, although equids are not considered reservoirs of Brucella species, once infected, they may potentially transmit the pathogen to other species or even to humans, representing a public health concern. References ACHA, P. N.; SZYFRES, B. Zoonosis y enfermidades transmisibles comunes al hombre y a los animales. 3 th ed. Washington: Organización Panamericana de la Salud, p. AGUIAR, D. M.; CAVALCANTE, G. T.; LARA, M. C. C. S. H.; VILLALOBOS, E. M. C.; CUNHA, E. M. S.; OKUDA, L. H.; STÉFANO, E.; NASSAR, A. F. C.; SOUZA, G. O.; VASCONCELLOS, S. A.; LABRUNA, M. B.; CAMARGO, L. M. A.; GENNARI, S. M. Prevalência de anticorpos contra agentes virais ebacterianos em equídeos do Município de Monte Negro, Rondônia, Amazônia Ocidental Brasileira. Brazilian Journal of Veterinary Research and Animal Science, São Paulo, v. 45, n. 4, p , ALMEIDA, C. A. S.; RIBEIRO, M. G.; MEGID, J.; VASCONCELLOS, A. S.; BORGES, A. S.; BONESI, G. Ocorrência de aglutininas anti Brucella abortus em soros de equideos de abatedouro. In: CONGRESSO NACIONAL DE SAÚDE PÚBLICA VETERINÁRIA, 2., 2007, Fortaleza. Anais... Fortaleza: CNSPV, CD- ROM. ALMEIDA, F. Q.; SILVA, V. P. Progresso cientifico em equideocultura na 1ª década do século XXI. Revista Brasileira de Zootecnia, Piracicaba, v. 39, p , Suplemento. ANTUNES, J. M. A. P.; ALLENDORF, S. D.; APPOLINÁRIO, C. M.; PERES, M. G.; PEROTTA, J. H.; NEVES, T. B.; DECONTO, I.; BARROS FILHO, I. R.; BIONDO, A. W.; MEGID, J. Serology for Brucella abortus in cart horses from an urban area in Brazil. Arquivo Brasileiro Medicina Veterinária e Zootecnia, Belo Horizonte, v. 65, n. 2, p , ARAÚJO, R. R.; PENA, L. J.; PENA, D. A.; DIAS, F. M.; MORAES, M. P. Ocorrência de anticorpos anti Brucella spp. em equídeos da região da zona da mata do estado de Minas Gerais, Brasil. Arquivos do Instituto Biológico. São Paulo, v. 76, n. 4, p ,

8 Motta, R. G. et al. ARRUDA, F. R.; SILVA, M. H.; SOARES FILHO, P. M.; CAMPOS, A. C.; AZEVEDO, E. O. Brucelose equina no Estado da Paraíba. Medicina Veterinária, Recife, v. 6, n. 1, p. 7-10, BERTU, W. J.; OCHOLI, R. A.; GUSI, A. M.; ABDULLAHI, S.; ZWANDOR, N. J.; DURBI, I. A. A.; OPARA, J.; OKEWOLE, P. A. Brucella abortus infection in a multispecies livestock farm in Nigeria. International Journal of Biotechnology and Food Science, Kobe, v. 3, n. 3, p , CARRAZZA, L. G.; JUNQUEIRA, Y. F.; CARRAZZA, T. G.; OLIVEIRA, P. R.; LIMA-RIBEIRO, A. M. C. Soroepidemiologia da brucelose em equinos de tração em áreas urbanas no município de Uberlândia-MG. Horizonte Cientifico, Uberlândia, v. 4, n. 2, p. 1-18, CHAVES, D. P.; BRITO, D. R. B.; SANTOS, A. C. G.; VAZ, J. F. R.; ANUNCIAÇÃO, A. R. Soroprevalência de mormo, anemia infecciosa equina e brucelose do cavalo baixadeiro. Revista Brasileira de Ciência Veterinária, Rio de Janeiro, v. 22, n. 1, p , COHEN, N. D.; CARTER, G. K.; MULLAN, W. C. Fistulous withers in horses. Journal of the American Veterinary Medical Association, Schaumburg, v. 201, n. 1, p , DORNELES, E. M. S.; FERNANDES, L. G.; SANTANA, J. A.; FREITAS, F. J. C.; LIMA, J. M.; BARROS, I. O.; SAKAMOTO, S. M.; HEINEMANN, M. B.; LAGE, A. P. Anti-Brucella abortus antibodies in free-ranging equids from Mossoró, Rio Grande do Norte, Brazil. Semina: Ciências Agrárias, Londrina, v. 34, n. 1, p , FEITOSA, M. H.; BITTAR, C. R.; GOMES, S. P. Brucelose: levantamento sorológico no estado de São Paulo no período de 1977 a Veterinária e Zootecnia, Botucatu, v. 3, n. 1, p. 9-15, FURQUIM, M. E. C.; SOUZA, M. A.; MAGALHÃES, L. F.; WILSON, T. M.; LIMA, A. M. C. Investigação da brucelose em equídeos abatidos em frigorífico exportador. Veterinária Notícias, Uberlândia, v. 18, n. 2, p , GONZÁLEZ, R. A.; GONZÁLEZ-REYES, I.; FLORES- GUTIÉRREZ, G. H. Prevalence of Brucella abortus antibodies in equines of a tropical region of México. Canadian Journal of Veterinary Research, Ottawa, v. 70, n. 1, p , INSTITUTO BRASILEIRO DE GEOGRAFIA ESTATÍSTICA IBGE. 9º Censo Agropecuário. Brasília: IBGE Produto, Disponível em: < gov.br/home>. Acesso em: 22 jul JARDIM, E. C.; ALMEIDA, M. M. R.; CÂNDIDA, M. F.; SILVA, R. L.; FICHTNER, S. S. Presença de aglutininas anti-brucella em equinos no Estado de Goiás. Pesquisa Agropecuária Tropical, Goiânia, v. 8, n. 1, p , JUNQUEIRA JÚNIOR, D. G.; DORNELES, E. M. S.; GONÇALVES, V. S. P.; SANTANA, J. A.; ALMEIDA, V. M. A.; NICOLINO, R. R. Brucellosis in working equines of cattle farms from Minas Gerais state, Brazil. Preventive Veterinary Medicine, Geneva, v. 121, n. 1, p , KARTHIK, K.; PRABAKAR, G.; BHARATHI, R.; KHURANA, S. K.; DHAMA, K. Equine brucellosis: review on epidemiology, pathogenesis, clinical signs, prevention and control. Journal of Experimental Biology and Agricultural Sciences, Switzerland, v. 4, n. 4, p , LANGONI, H.; SILVA, A. V. Comportamento sorológico de aglutininas anti-brucella em soro de equídeos. Revista Brasileira de Medicina Veterinária, Rio de Janeiro, v. 19, n. 1, p , LOPES, C. F. A.; MOLNÁR, L.; MOLNÁR, E. Avaliação soroepidemiológica da brucelose em animais e humanos procedentes da zonabragantina no Estado do Pará, Brasil. Revista Brasileira de Reprodução Animal, Belo Horizonte, v. 23, n. 1, p , MEGID, J.; RIBEIRO, M. G.; PAES, A. C. Doenças infecciosas em animais de produção e companhia. Rio de Janeiro: Editora Roca, p. NARDI JÚNIOR, G..; MEGID, J.; VICENTE, A. F.; LISTONI, F. J. P.; MONTEIRO, F. M.; LARA, G. H. B.; MOTTA, R. G.; CHACUR, M. G. M.; RIBEIRO, M. G. Comparison of Brucella agar, CITA and Farrell media for selective isolation of Brucella abortus from semen of bovine bulls. African Journal of Microbiology Research, Lagos, v. 9, n. 9, p , NIELSEN, K.; SMITH, P.; WIDDISON, J.; GALL, D.; KELLY, L.; KELLY, W.; NICOLETTI, P. Serological relationship between cattle exposed to Brucella abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7. Veterinary Microbiology, Victoria, v. 100, n. 1, p , OFFICE INTERNATIONAL DES EPIZOOTIES OIE. Brucellosis. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 7 th ed. Paris: World Organisation for Animal Health, Available at: < int/manual-of-diagnostic-tests-andvaccines-for-terrestrialanimals>. Accessed at: 25 july

9 Serodiagnosis of brucellosis in mules from Southwestern Goiás State, Brazil OLIVEIRA FILHO, R. B.; MALTA, K. C.; SANTANA, V. L. A.; SOUZA, M. M. A.; STIPP, D. T.; PINHEIRO JÚNIOR, J. W. Prevalência da infecção por Brucella spp. em equídeos na microrregião do Brejo Paraibano. Veterinária e Zootecnia, Botucatu, v. 20, n. 4, p , OLIVEIRA, Q. C.; MOREIRA, V. S.; LIMA, C. S. Brucelose em equinos. Revista de Medicina, São Paulo, v. 9, n. 1, p , OMER, M. K.; SKEJERVE, E.; HOLSTAD, G.; WOLDEHIWET, Z.; MACMILLAN, A. P. Prevalence of antibodies to Brucella spp. in cattle, sheep, goats, horses and camels in the State of Eritrea; influence of husbandry systems. Epidemiology and Infection, Cambridge, v. 125, n. 2, p , PINHO, A. P. V. B.; KURODA, R. B. S.; VASCONCELLOS, S. A.; FERREIRA NETO, J. S.; OSSADA, R.; SOUZA, V. A. F.; ROCHA, K. S.; PAZ, G. S.; MORAES, C. C. G. Estudo sorológico da brucelose e leptospirose em equídeos da ilha de Maiandeua (Algodoal) no Estado do Pará. Semina: Ciências Agrárias, Londrina, v. 35, n. 6, p , POESTER, F. P.; GONÇALVES, V. S. P.; LAGE, A. P. Brucellosis in Brazil. Veterinary Microbiology, Geneva, v. 90, n. 1, p , QUINN, P. J.; MARKEY, B.; CARTER, M. E.; DONNELLY, W. J.; LEONARD, E. C. Microbiologia veterinária e doenças infecciosas. 2 th ed. Porto Alegre: Artmed, p. RADOSTITS, O. M.; GAY, C. C.; HINCHCLIFF, K. W.; CONSTABLE, P. D. Veterinary medicine: a textbook of the diseases of cattle, horses, sheep, pigs, and goats. 10 th ed. Philadelphia: Saunders, p. RIBEIRO, M. G.; MOTTA, R. G.; ALMEIDA, A. A. S. Brucelose equina: aspectos da doença no Brasil. Revista Brasileira de Reprodução Animal, Belo Horizonte, v. 32, n. 2, p , RIBEIRO, M. G.; NARDI JUNIOR, G.; MEGID, J.; PAES, A. C.; LISTONI, F. J. P. Anti-Brucella abortus agglutinins in serum and secretion of fistulous withers in horses. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, Belo Horizonte, v. 55, n. 1, p , SANTOS, A. L. Q.; LIMA-RIBEIRO, A. M. C.; OLIVEIRA, S. R. P.; KAMINISHI, A. P. Z.; ANDRADE, M. B.; MENEZES, L. T.; SOUZA, R. R.; FERREIRA, C. H.; NASCIMENTO, L. R.; MORAES, F. M. Soroepidemiologia da Brucelose em equinos de trabalho de áreas rurais do Município de Uberlândia-MG. Publicações em Medicina Veterinária PUVET, Londrina, v. 6, n. 12, p , SANTOS, R. F.; SILVA, G. C. P.; ASSIS, N. A.; MATHIAS, L. A. Brucella spp. in equines slaughtered in the south region of Brazil. Arquivo Brasileiro de Medicina Veterinária e Zootecnia, Belo Horizonte, v. 68, n. 4, p , SILVA, L. A. F.; ACYPRESTE, C. S.; EURIDES, D.; MACHADO, G. V.; DIAS-FILHO, F. C.; FIORAVANTI, M. C. S.; RAMOS, L. S. Prevalência da Brucelose em equinos com bursite cervical ou nucal. Arquivos de Ciências Veterinárias e Zoologia da UNIPAR, Umuarama, v. 4, n. 1, p , SILVA, L. A. F.; BARBOSA, V. T.; SOARES, L. S.; LIMA, C. R. O.; COELHO, C. M. M.; LIMA, I. R.; ESTEVES, G. I. F.; ARAÚJO, I. F. L.; COSTA, G. L.; FRANCO, L. G.; MOURA, M. I.; SILVA, M. A. M. Brucelose em equino portador de bursite fistulosa de cernelha relato de caso. Veterinária. Notícias, Uberlândia, v. 12, n. 2, p , STARK, C. B.; AMARAL, L. A.; HENTGES, A.; JORGE, S.; MARTINS, P. L.; BANDEIRA, F. S.; FERNANDES, C. P. H.; NOGUEIRA, C. E. W.; BROD, C. S.; RECUERO, A. L. C. Pesquisa de anticorpos anti-brucela em animais de tração atendidos no hospital veterinário da Universidade Federal de Pelotas. In: CONGRESSO BRASILEIRO DE MEDICINA VETERINÁRIA, 35., 2008, Gramado. Anais... Gramado: Conbravet, Disponível em: < conbravet2008/anais/cd/resumos/r pdf>. Acesso em: 27 jul TRIOLA, M. F. Introdução à estatística: atualização da tecnologia. 11 th ed. Rio de Janeiro: LTC, p. VIANA, F. C.; REIS, R.; SANTOS, W. L. M. Inquérito sorológico para brucelose equina em Minas Gerais. Arquivo da Escola Superior de Veterinária do Estado de Minas Gerais, Belo Horizonte, v. 33, n. 2, p ,

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