RESUMO
Brucellosis is a prevalent disease in Costa Rica (CR), with an increasing number of human infections. Close to half of homes in CR have one or more dogs, corresponding to â¼1.4 million canines, most of them in the Central Valley within or near the cities of San José, Heredia, and Alajuela. From 302 dog sera collected from this region, 19 were positive for Brucella canis antigens, and five had antibodies against smooth lipopolysaccharide, suggesting infections by both B. canis and other Brucella species. B. canis strains were isolated in the Central Valley from 26 kennel dogs and three pet dogs, all displaying clinical signs of canine brucellosis. We detected three recent introductions of different B. canis strains in kennels: two traced from Mexico and one from Panama. Multiple locus-variable number tandem repeats (MLVA-16) and whole-genome sequencing (WGSA) analyses showed that B. canis CR strains comprise three main lineages. The tree topologies obtained by WGSA and MLVA-16 just partially agreed, indicating that the latter analysis is not suitable for phylogenetic studies. The fatty acid methyl ester analysis resolved five different B. canis groups, showing less resolution power than the MLVA-16 and WGSA. Lactobacillic acid was absent in linages I and II but present in linage III, supporting the recent introductions of B. canis strains from Mexico. B. canis displaying putative functional cyclopropane synthase for the synthesis of lactobacillic acid are phylogenetically intertwined with B. canis with non-functional protein, indicating that mutations have occurred independently in the various lineages.
Assuntos
Brucella canis/genética , Brucelose/epidemiologia , Brucelose/veterinária , Surtos de Doenças/veterinária , Doenças do Cão/microbiologia , Filogenia , Animais , Brucella canis/classificação , Brucella canis/patogenicidade , Costa Rica/epidemiologia , Doenças do Cão/epidemiologia , Cães , Evolução Molecular , Feminino , Variação Genética , Genoma Bacteriano , Genótipo , Espécies Introduzidas , Masculino , México , Panamá , Animais de Estimação/microbiologia , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: Brucella canis is the etiological agent of canine brucellosis, a worldwide neglected zoonosis that constitutes one of the major infectious causes of infertility and reproductive failure in dogs. Although genomic information available for this pathogen has increased in recent years, here we report the first genome sequencing of a B. canis strain in Chile, and the differences in virulence genes with other B. canis strains. RESULTS: Genome assembly produced a total length of 3,289,216 bp, N50 of 95,163 and GC% of 57.27, organized in 54 contigs in chromosome I, and 21 contigs in chromosome II. The genome annotation identified a total of 1981 CDS, 3 rRNA and 36 tRNA in chromosome I, and 1113 CDS and 10 tRNA in chromosome II. There is little variation between the different strains and the SCL isolate. Phylogenetic analysis showed that the Chilean SCL strain is closely related to B. canis and B. suis strains. Small differences were found when compared to the Serbian isolate, but all strains shared the same recent common ancestor. Finally, changes in the sequence of some virulence factors showed that the SCL strain is similar to other South American B. canis strains. CONCLUSIONS: This work sequenced and characterized the complete genome of B. canis strain SCL, evidencing the complete presence of all the genes of the virB operon, and minor changes in outer membrane proteins and in the urease operon. Our data suggest that B. canis was introduced from North America and then spread throughout the South American continent.
Assuntos
Animais , Cães , Brucelose/epidemiologia , Brucella canis/genética , Brucella canis/patogenicidade , Urease/genética , Brucelose/transmissão , Zoonoses , Chile , GenomaRESUMO
Canine brucellosisis an infectious disease caused by bacteria of the genus Brucella, with world wide distribution and zoonotic impact, and in humans and animals is a neglected disease. In the present study, the sero prevalence of B. canis and B. abortus were determined in a probabilistic sample of housed dogs from the Atlantic Rainforest area of the state of Paraíba, Brazil, and the factors associated with sero positivity. A total of 386 dogs over three months of age were used. For the search for anti-B.canis antibodies the agar gel immune diffusion test (IDGA) was used as a screening and IDGA+2ME as confirmatory test, and to search for anti-B. abortus antibodies the Rose Bengal test (RBT) test was used. Apparent and real prevalences were calculated, and robust Poisson regression was used to identify factors associated with prevalence. The real prevalence fB. Canis was 12.6% and of B. abortus was 22.8%. The factors associated with sero positivity for B. canis were age greater than 10 years (prevalence ratio; PR = 6.38; P = 0.024) and dogs reared in they ard (PR = 5.20; P = 0.035) and for B. abortus was no treplacement of water of animals everyday (PR = 1.48; P = 0.033). It can be concluded that the prevalence of B. canis and B. Abortus in the region is high, which warns to the adopting of control and prevention measures, as well as greater care in the management of animals, especially for elderly dogs.(AU)
A brucelose canina é uma doença infecciosa causada por bactérias do gênero Brucella, com distribuição mundial e de caráter zoonótico, e em humanos e animais é uma doença negligenciada. No presente estudo foram determinados as soroprevalências de B. canis e B. abortusem uma amostra probabilística de cães domiciliados da área urbana de oito municípios localizados na região da Mata Atlântica do Estado da Paraíba, Brasil, e os fatores associados com a soropositividade. Foram utilizados 386 cães com mais de três meses de idade. Para a pesquisa de anticorpos anti-B. canis foi utilizado o teste de imunodifusão em gel de ágar (IDGA) como triagem e IDGA+2ME como confirmatório, e para a pesquisa de anticorpos anti-B. abortusfoi utilizado o teste do antígeno acidificado tamponado (AAT). Foram calculadas as prevalências aparente e real, e para a identificação de fatores associados com a prevalência foi empregada regressão robusta de Poisson. A prevalência real de B. canis foi de 12,6% e de B. abortusfoi 22,8%. Os fatores associados com a soropositividade para B. canis foram idade maior que 10 anos (razão de prevalência; RP = 6,38; P = 0,024) e cães criados presos no quintal (RP = 5,20; P = 0,035) e para B. abortus foi não trocar a água dos animais todos os dias (RP = 1,48; P = 0,033). Conclui-se que a prevalência de B. canis e B. abortusem cães da região é alta, o que alerta para a necessidade de adoção de medidas de controle e prevenção, bem como são sugeridos maiores cuidados no manejo dos animais, sobretudo cães idosos.(AU)
Assuntos
Animais , Cães , Brucella abortus/patogenicidade , Brucelose/epidemiologia , Brucelose/prevenção & controle , Brucelose/veterinária , Brucella canis/patogenicidadeRESUMO
BACKGROUND/OBJECTIVES: Vaccination is the most important tool for controlling brucellosis, but currently there is no vaccine available for canine brucellosis, which is a zoonotic disease of worldwide distribution caused by Brucella canis. This study aimed to evaluate protection and immune response induced by Brucella ovis ΔabcBA (BoΔabcBA) encapsulated with alginate against the challenge with Brucella canis in mice and to assess the safety of this strain for dogs. METHODS: Intracellular growth of the vaccine strain BoΔabcBA was assessed in canine and ovine macrophages. Protection induced by BoΔabcBA against virulent Brucella canis was evaluated in the mouse model. Safety of the vaccine strain BoΔabcBA was assessed in experimentally inoculated dogs. RESULTS: Wild type B. ovis and B. canis had similar internalization and intracellular multiplication profiles in both canine and ovine macrophages. The BoΔabcBA strain had an attenuated phenotype in both canine and ovine macrophages. Immunization of BALB/c mice with alginate-encapsulated BoΔabcBA (108 CFU) induced lymphocyte proliferation, production of IL-10 and IFN-γ, and protected against experimental challenge with B. canis. Dogs immunized with alginate-encapsulated BoΔabcBA (109 CFU) seroconverted, and had no hematologic, biochemical or clinical changes. Furthermore, BoΔabcBA was not detected by isolation or PCR performed using blood, semen, urine samples or vaginal swabs at any time point over the course of this study. BoΔabcBA was isolated from lymph nodes near to the site of inoculation in two dogs at 22 weeks post immunization. CONCLUSION: Encapsulated BoΔabcBA protected mice against experimental B. canis infection, and it is safe for dogs. Therefore, B. ovis ΔabcBA has potential as a vaccine candidate for canine brucellosis prevention.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Vacina contra Brucelose/imunologia , Brucella ovis/genética , Brucelose/prevenção & controle , Doenças do Cão/prevenção & controle , Alginatos/química , Animais , Formação de Anticorpos , Brucella canis/patogenicidade , Brucella ovis/imunologia , Brucella ovis/isolamento & purificação , Brucelose/microbiologia , Brucelose/patologia , Doenças do Cão/microbiologia , Doenças do Cão/patologia , Cães , Feminino , Imunização , Fígado/microbiologia , Fígado/fisiologia , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , OvinosRESUMO
Brucella canis infection is an underdiagnosed zoonotic disease. Knowledge about perinatal brucellosis in dogs is extremely limited, although foetuses and neonates are under risk of infection due to vertical transmission. In this study, immunohistochemistry was used to determine tissue distribution and cell tropism of B. canis in canine foetuses and neonates. Diagnosis of B. canis in tissues of naturally infected pups was based on PCR and sequencing of amplicons, bacterial isolation, and immunohistochemistry, whose specificity was confirmed by laser capture microdissection. PCR positivity among 200 puppies was 21%, and nine isolates of B. canis were obtained. Tissues from 13 PCR-positive puppies (4 stillborn and 9 neonates) presented widespread immunolabeling. Stomach, intestines, kidney, nervous system, and umbilicus were positive in all animals tested. Other frequently infected organs included the liver (92%), lungs (85%), lymph nodes (69%), and spleen (62%). Immunolabeled coccobacilli occurred mostly in macrophages, but they were also observed in erythrocytes, epithelial cells of gastrointestinal mucosa, renal tubules, epidermis, adipocytes, choroid plexus, ependyma, neuroblasts, blood vessels endothelium, muscle cells, and in the intestinal lumen. These results largely expand our knowledge about perinatal brucellosis in the dog, clearly demonstrating a pantropic distribution of B. canis in naturally infected foetuses and neonates.
Assuntos
Brucella canis/isolamento & purificação , Brucelose/veterinária , Doenças do Cão/epidemiologia , Tropismo/fisiologia , Zoonoses/epidemiologia , Animais , Animais Recém-Nascidos , Brasil/epidemiologia , Brucella canis/classificação , Brucella canis/genética , Brucella canis/patogenicidade , Brucelose/epidemiologia , Brucelose/microbiologia , Brucelose/patologia , Doenças do Cão/microbiologia , Doenças do Cão/patologia , Cães , Feminino , Feto , Microdissecção e Captura a Laser , Fígado/microbiologia , Pulmão/microbiologia , Linfonodos/microbiologia , Macrófagos/microbiologia , Masculino , Reação em Cadeia da Polimerase , Baço/microbiologia , Zoonoses/microbiologia , Zoonoses/patologiaRESUMO
Brucella canis is a facultative intracellular bacterium responsible for brucellosis in dogs, a chronic and zoonotic infectious disease endemic in Brazil, which promotes reproductive failure and involvement of the lymphoid and articular systems. The infection has been commonly described in breeding kennels, being difficult to treat and control. This study reports an outbreak of brucellosis in a commercial kennel in Guarulhos, SP, Brazil, comprised of 32 dogs (four males and 28 females) from five different breeds, where episodes of abortion had been happening since October 2015. Brucellosis was considered a potential cause of reproductive impairment only a year and a half after the first episode. Anamnesis, clinical examination and serological and bacteriological tests were conducted in all dogs. Of the 32 dogs, 28 had at least one clinical sign compatible with brucellosis, with increased lymph node volume and abortion being the most frequently observed signs. Twenty-six dogs had positive results in at least one of the laboratory tests used for the diagnosis, indicating the infections prevalence to be 81.25% in the kennel. Regarding microbiological diagnosis, B. canis was isolated in blood samples of 22 dogs, in vaginal swabs of nine females and in semen or preputial swabs of two males. Twenty-three dogs had positive results in the serological test.
A Brucella canis é uma bactéria intracelular facultativa responsável pela brucelose nos cães, uma doença infectocontagiosa de caráter crônico e zoonótico, endêmica no Brasil e associada a problemas reprodutivos e comprometimento dos sistemas linfoide e articular. A infecção é comumente descrita em canis de reprodutores, sendo de difícil tratamento e controle. Este trabalho relata um surto de brucelose em um canil comercial localizado em Guarulhos (São Paulo), composto por 32 cães (4 machos e 28 fêmeas) de 5 raças distintas, no qual episódios de abortamento foram relatados desde outubro de 2015. A suspeita de brucelose foi aventada apenas um ano e meio após a ocorrência do primeiro episódio de abortamento, sendo realizados anamnese, exames clínicos e diagnósticos sorológico e bacteriológico em todos os cães do plantel. Dos 32 cães, 28 apresentaram pelo menos um sinal clínico compatível com brucelose, sendo o aumento de linfonodos e o abortamento os sinais mais frequentemente observados. Vinte e seis animais apresentaram resultado positivo em pelo menos um dos testes laboratoriais usados para o diagnóstico, indicando prevalência de 81,25% na criação. No diagnóstico microbiológico, houve isolamento de B. canis em amostras de sangue de 22 cães, em amostras de swab vaginal de 9 fêmeas e em sêmen ou swab prepucial de 2 machos.
Assuntos
Animais , Cães , Brucelose/complicações , Brucelose/diagnóstico , Brucelose/veterinária , Cães/anormalidades , Aborto Animal , Brucella canis/patogenicidadeRESUMO
Brucella canis is a facultative intracellular bacterium responsible for brucellosis in dogs, a chronic and zoonotic infectious disease endemic in Brazil, which promotes reproductive failure and involvement of the lymphoid and articular systems. The infection has been commonly described in breeding kennels, being difficult to treat and control. This study reports an outbreak of brucellosis in a commercial kennel in Guarulhos, SP, Brazil, comprised of 32 dogs (four males and 28 females) from five different breeds, where episodes of abortion had been happening since October 2015. Brucellosis was considered a potential cause of reproductive impairment only a year and a half after the first episode. Anamnesis, clinical examination and serological and bacteriological tests were conducted in all dogs. Of the 32 dogs, 28 had at least one clinical sign compatible with brucellosis, with increased lymph node volume and abortion being the most frequently observed signs. Twenty-six dogs had positive results in at least one of the laboratory tests used for the diagnosis, indicating the infections prevalence to be 81.25% in the kennel. Regarding microbiological diagnosis, B. canis was isolated in blood samples of 22 dogs, in vaginal swabs of nine females and in semen or preputial swabs of two males. Twenty-three dogs had positive results in the serological test.(AU)
A Brucella canis é uma bactéria intracelular facultativa responsável pela brucelose nos cães, uma doença infectocontagiosa de caráter crônico e zoonótico, endêmica no Brasil e associada a problemas reprodutivos e comprometimento dos sistemas linfoide e articular. A infecção é comumente descrita em canis de reprodutores, sendo de difícil tratamento e controle. Este trabalho relata um surto de brucelose em um canil comercial localizado em Guarulhos (São Paulo), composto por 32 cães (4 machos e 28 fêmeas) de 5 raças distintas, no qual episódios de abortamento foram relatados desde outubro de 2015. A suspeita de brucelose foi aventada apenas um ano e meio após a ocorrência do primeiro episódio de abortamento, sendo realizados anamnese, exames clínicos e diagnósticos sorológico e bacteriológico em todos os cães do plantel. Dos 32 cães, 28 apresentaram pelo menos um sinal clínico compatível com brucelose, sendo o aumento de linfonodos e o abortamento os sinais mais frequentemente observados. Vinte e seis animais apresentaram resultado positivo em pelo menos um dos testes laboratoriais usados para o diagnóstico, indicando prevalência de 81,25% na criação. No diagnóstico microbiológico, houve isolamento de B. canis em amostras de sangue de 22 cães, em amostras de swab vaginal de 9 fêmeas e em sêmen ou swab prepucial de 2 machos.(AU)
Assuntos
Animais , Cães , Cães/anormalidades , Brucelose/complicações , Brucelose/diagnóstico , Brucelose/veterinária , Brucella canis/patogenicidade , Aborto AnimalRESUMO
Canine brucellosis caused by Brucella canis is a disease of dogs and a zoonotic risk. B. canis harbors most of the virulence determinants defined for the genus, but its pathogenic strategy remains unclear since it has not been demonstrated that this natural rough bacterium is an intracellular pathogen. Studies of B. canis outbreaks in kennel facilities indicated that infected dogs displaying clinical signs did not present hematological alterations. A virulent B. canis strain isolated from those outbreaks readily replicated in different organs of mice for a protracted period. However, the levels of tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-12 in serum were close to background levels. Furthermore, B. canis induced lower levels of gamma interferon, less inflammation of the spleen, and a reduced number of granulomas in the liver in mice than did B. abortus. When the interaction of B. canis with cells was studied ex vivo, two patterns were observed, a predominant scattered cell-associated pattern of nonviable bacteria and an infrequent intracellular replicative pattern of viable bacteria in a perinuclear location. The second pattern, responsible for the increase in intracellular multiplication, was dependent on the type IV secretion system VirB and was seen only if the inoculum used for cell infections was in early exponential phase. Intracellular replicative B. canis followed an intracellular trafficking route undistinguishable from that of B. abortus. Although B. canis induces a lower proinflammatory response and has a stealthier replication cycle, it still displays the pathogenic properties of the genus and the ability to persist in infected organs based on the ability to multiply intracellularly.
Assuntos
Brucella abortus/imunologia , Brucella canis/imunologia , Brucelose/imunologia , Sistemas de Secreção Tipo IV/imunologia , Animais , Medula Óssea/imunologia , Medula Óssea/patologia , Brucella abortus/crescimento & desenvolvimento , Brucella abortus/patogenicidade , Brucella canis/crescimento & desenvolvimento , Brucella canis/patogenicidade , Brucelose/genética , Brucelose/patologia , Cães , Feminino , Expressão Gênica , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Fígado/imunologia , Fígado/patologia , Linfonodos/imunologia , Linfonodos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade da Espécie , Baço/imunologia , Baço/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Sistemas de Secreção Tipo IV/genéticaRESUMO
Inhalation is a common route for Brucella infection. We investigated whether Brucella species can invade and replicate within alveolar(A549) and bronchial (Calu-6 and 16HBE14o-) human epithelial cells. The number of adherent and intracellular bacteria was higher for rough strains (Brucella canis and Brucella abortus RB51) than for smooth strains (B. abortus 2308 and Brucella suis 1330). Only smooth strains exhibited efficient intracellular replication (1.5-3.5 log increase at 24 h p.i.). A B. abortus mutant with defective expression of the type IV secretion system did not replicate. B. abortus internalization was inhibited by specific inhibitors of microfilaments, microtubules and PI3-kinase activity. As assessed with fluorescent probes, B. abortus infection did not affect the viability of A549 and 16HBE14o- cells, but increased the percentage of injured cells (both strains) and dead cells (RB51) in Calu-6 cultures. LDH levels were increased in supernatants of Calu-6 and 16HBE14o- cells infected with B. abortus RB51, and to a lower extent in Calu-6 infected with B. abortus 2308. No apoptosis was detected by TUNEL upon infection with smooth or rough B. abortus. This study shows that smooth brucellae can infect and replicate in human respiratory epithelial cells inducing minimal or null cytotoxicity.
Assuntos
Brucella abortus/patogenicidade , Brucella canis/patogenicidade , Brucella suis/patogenicidade , Morte Celular , Células Epiteliais/microbiologia , Aderência Bacteriana , Linhagem Celular , Sobrevivência Celular , Citoplasma/microbiologia , HumanosRESUMO
The less mucoid strain of Brucella canis or M- strain is used for the serologic diagnosis of canine brucellosis. While this strain is avirulent in dogs, we report the case of clinical brucellosis that developed in a laboratory worker a few days after handling live M- cells for antigen production.