RESUMO
Brucella abortus induces an inflammatory response that stimulates the endocrine system resulting in the secretion of cortisol and dehydroepiandrosterone (DHEA). Osteoarticular brucellosis is the most common presentation of the active disease in humans, and we have previously demonstrated that B. abortus infection inhibits osteoblast function. We aimed to evaluate the role of cortisol and DHEA on osteoblast during B. abortus infection. B. abortus infection induces apoptosis and inhibits osteoblast function. DHEA treatment reversed the effect of B. abortus infection on osteoblast by increasing their proliferation, inhibiting osteoblast apoptosis, and reversing the inhibitory effect of B. abortus on osteoblast differentiation and function. By contrast, cortisol increased the effect of B. abortus infection. Cortisol regulates target genes by binding to the glucocorticoid receptor (GR). B. abortus infection inhibited GRα expression. Cell responses to cortisol not only depend on GR expression but also on its intracellular bioavailability, that is, dependent on the activity of the isoenzymes 11ß-hydroxysteroid dehydrogenase (HSD) type-1, 11ß-HSD2 (which convert cortisone to cortisol and vice versa, respectively). Alterations in the expression of these isoenzymes in bone cells are associated with bone loss. B. abortus infection increased 11ß-HSD1 expression but had no effect on 11ß-HSD2. DHEA reversed the inhibitory effect induced by B. abortus infection on osteoblast matrix deposition in an estrogen receptor- and ERK1/2-dependent manner. We conclude that DHEA intervention improves osteoblast function during B. abortus infection making it a potential candidate to ameliorate the osteoarticular symptoms of brucellosis.
Assuntos
Brucella abortus/fisiologia , Brucelose Bovina/metabolismo , Brucelose Bovina/microbiologia , Desidroepiandrosterona/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteoblastos/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Apoptose , Biomarcadores , Brucelose Bovina/genética , Brucelose Bovina/patologia , Bovinos , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Expressão Gênica , Camundongos , Viabilidade Microbiana , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismoRESUMO
Brucellosis is one of the most important zoonotic diseases in the world. Considering its strict zoonotic nature, understanding of the pathogenesis and immunity of Brucella spp. in natural animal hosts is essential to prevent human infections. Natural resistance against brucellosis has been demonstrated in cattle, and it is associated with the ability of macrophages to prevent intracellular replication of Brucella abortus. Identification of breeds that are resistant to B. abortus may contribute for controlling and eradicating brucellosis in cattle. This study aimed to compare macrophages from Nelore (Bos taurus indicus) or Holstein (Bos taurus taurus) regarding their resistance to B. abortus infection. Macrophages from Nelore were significantly more efficient in controlling intracellular growth of B. abortus when compared to Holstein macrophages even under intralysosomal iron restricting conditions. Furthermore, Nelore macrophages had higher transcription levels of inducible nitric oxide synthase (iNOS) and TNF-α at 12h post-infection (hpi) and higher levels of IL-12 at 24 hpi when compared to Holstein macrophages. Conversely, Holstein macrophages had higher levels of IL-10 transcripts at 24 hpi. Macrohages from Nelore also generated more nitric oxide (NO) in response to B. abortus infection when compared to Holstein macrophages. In conclusion, cultured Nelore macrophages are more effective in controlling intracellular replication of B. abortus, suggesting that Nelore cattle is likely to have a higher degree of natural resistance to brucellosis than Holstein.
Assuntos
Brucella abortus/imunologia , Brucelose Bovina/imunologia , Bovinos/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Administração através da Mucosa , Animais , Brucella abortus/patogenicidade , Brucella abortus/ultraestrutura , Brucelose Bovina/genética , Brucelose Bovina/metabolismo , Bovinos/genética , Bovinos/microbiologia , Humanos , Imunidade Inata , Interleucina-10/genética , Interleucina-12/genética , Ferro/metabolismo , Macrófagos/metabolismo , Masculino , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da Espécie , Transcriptoma , Fator de Necrose Tumoral alfa/genética , ZoonosesRESUMO
This paper aimed to determine the excretion period of B19 vaccine strain during a complete reproductive cycle (from estrus synchronization, artificial insemination, pregnancy and until 30 days after parturition) of dairy cows from 3 to 9 years old that were previously vaccinated from 3 to 8 months. Three groups were monitored with monthly milk and urine collection during 12 months: G1 with seven cows from 3 to 4 years old; G2 with three cows from 5 to 6 years old; and G3 with four cows from 7 to 9 years old. Urine and milk samples were submitted to bacteriological culture and urine and PCR reactions for detection of Brucella spp. and PCR-multiplex for B19 strain identification. Ring test (RT) was also performed in the milk samples, and serum samples were tested by buffered acidified plate antigen test (BAPA). All animals were serologically negative at BAPA and Brucella spp. was not isolated from both urine and milk samples. RT revealed 13/210 (6.2%) positive milk samples. PCR reactions detected DNA of Brucella spp. in 86/420 (20.5%) samples. In urine it was found a significantly higher frequency (35.2%; 74/210) than in milk (5.7%; 12/210), more frequently from the estrus to 150 days of pregnancy and after parturition (6.7%; 10/150), and from 150 days of pregnancy to parturition (3.4%; 2/60), and they were all identified as B19 strain. In three groups, intermittent excretion of B19 strain was detected mainly in urine samples, which confirmed its multiplication and persistence in cows for until 9 years.
Assuntos
Animais , Bovinos , Brucelose Bovina/genética , Técnicas In Vitro , Reação em Cadeia da Polimerase/métodos , Sincronização do Estro/métodos , Vacina contra Brucelose/genética , Amostras de Alimentos , Métodos , Testes SorológicosRESUMO
Brucellosis is a worldwide zoonotic infectious disease that has a significant economic impact on animal production and human public health. We characterized the gene expression profile of B. abortus-infected monocyte-derived macrophages (MDMs) from naïve cattle naturally resistant (R) or susceptible (S) to brucellosis using a cDNA microarray technology. Our data indicate that (1) B. abortus induced a slightly increased genome activation in R MDMs and a down-regulated transcriptome in S MDMs, during the onset of infection, (2) R MDMs had the ability to mount a type 1 immune response against B. abortus infection which was impaired in S cells, and (3) the host cell activity was not altered after 12 h post-B. abortus infection in R MDMs while the cell cycle was largely arrested in infected S MDMs at 12 h p.i. These results contribute to an improved understanding of how host responses may be manipulated to prevent infection by brucellae.
Assuntos
Brucella abortus/imunologia , Brucelose Bovina/genética , Suscetibilidade a Doenças/veterinária , Imunidade Inata , Macrófagos , Animais , Brucelose Bovina/imunologia , Bovinos , Suscetibilidade a Doenças/imunologia , Regulação para Baixo/imunologia , Perfilação da Expressão Gênica/veterinária , Análise de Sequência com Séries de Oligonucleotídeos/veterináriaRESUMO
The 3' untranslated region (3' UTR) of the bovine natural resistance-associated macrophage gene (NRAMP1 or SLC11A1) was genotyped in Colombian Creole Blanco Orejinegro (BON) (Bos taurus) (n = 140) and Zebu Brahman (Bos indicus) (Z) (n = 20) cattle and their crosses (BON x Zebu Brahman [B x Z] [n = 10]; Zebu Brahman x BON [Z x B] [n = 10]), and in animals from a Holstein x BON (H x B) (n = 10) cross. Direct sequencing and single-strand conformation polymorphism analysis (SSCP) helped in detecting the polymorphic behaviour. The association between resistance to brucellosis infection and SSCP genotype was evaluated using a macrophage in vitro killing assay employing a virulent Brucella abortus strain. The 3' UTR (GT) repeated polymorphism was gentoyped and its association with resistance to brucellosis was evaluated. When all breeds were grouped, a high frequency in the homozygote GT(12) (AA genotype) (0.823) and a very low frequency in the homozygote GT(10) (BB genotype) (0.047) were detected. The BON (0.963), Z x B (0.60) and H x B (1.00) cattle showed high GT(12) allele frequencies, unlike that seen for the B x Z and Zebu cattle (0.3002 and 0.218, respectively). The GT(10) allele was only found in the Zebu cattle (0.391). A significant association (p < 0.001) was found between the B. abortus macrophage in vitro killing assay phenotypes and the bovine SLC11A1 3' UTR genotypes, which suggests that the A allele may be associated with resistance. Because only nine animals had the BB genotype, the results require some confirmation in more extensive populations.
Assuntos
Regiões 3' não Traduzidas/química , Brucella abortus/patogenicidade , Proteínas de Transporte de Cátions/genética , Bovinos/genética , Polimorfismo Conformacional de Fita Simples , Animais , Brucelose Bovina/genética , Brucelose Bovina/imunologia , Cruzamentos Genéticos , Frequência do Gene , Marcadores Genéticos , Genótipo , Imunidade Inata/genética , Macrófagos/fisiologia , Repetições de Microssatélites , Análise de Sequência de DNARESUMO
The NRAMP1 gene encodes a divalent cation transporter, located in the phagolysosomal membrane of macrophages, that has been associated with resistance to intracellular pathogens. In cattle, natural resistance against brucellosis has been associated with polymorphisms at the 3' untranslated region (3'UTR) of the NRAMP1 gene, which are detectable by single-strand conformational analysis (SSCA). This study aimed to evaluate the association between NRAMP1 3'UTR polymorphisms and resistance against bovine brucellosis in experimental and natural infections. In experimentally infected pregnant cows, abortion occurred in 42.1% of cows with a resistant genotype (SSCA(r); n = 19) and in 43.1% of those with a susceptible genotype (SSCA(s); n = 23). Furthermore, no association between intensity of pathological changes and genotype was detected. In a farm with a very high prevalence of bovine brucellosis, the percentages of strains of the SSCA(r) genotype were 86 and 84% in serologically positive (n = 64) and negative (n = 36) cows, respectively. Therefore, no association was found between the NRAMP1-resistant allele and the resistant phenotype in either experimental or naturally occurring brucellosis. To further support these results, bacterial intracellular survival was assessed in bovine monocyte-derived macrophages from cattle with either the resistant or susceptible genotype. In agreement with our previous results, no difference was observed in the rates of intracellular survival of B. abortus within macrophages from cattle with susceptible or resistant genotypes. Taken together, these results indicate that these polymorphisms at the NRAMP1 3'UTR do not affect resistance against B. abortus in cattle and that they are therefore not suitable markers of natural resistance against bovine brucellosis.
Assuntos
Regiões 3' não Traduzidas/genética , Brucella abortus/classificação , Brucella abortus/patogenicidade , Brucelose Bovina/imunologia , Proteínas de Transporte de Cátions/genética , Polimorfismo Genético , Aborto Animal/genética , Aborto Animal/imunologia , Aborto Animal/microbiologia , Animais , Brucella abortus/genética , Brucelose Bovina/genética , Brucelose Bovina/microbiologia , Bovinos , Células Cultivadas , Feminino , Predisposição Genética para Doença , Genótipo , Macrófagos/microbiologia , Masculino , Doenças Placentárias/genética , Doenças Placentárias/imunologia , Doenças Placentárias/microbiologia , Doenças Placentárias/veterinária , Polimorfismo Conformacional de Fita Simples , GravidezRESUMO
Peripheral blood monocyte-derived macrophages were obtained from a herd of cows selected, bred, and confirmed as resistant or susceptible by in vivo challenge of Brucella abortus Strain 2308. The ability to control in vitro intracellular bacterial replication of B. abortus Strain 2308, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Montreal Strain 9003, Salmonella dublin Strain 5631, and Salmonella typhimurium Strain 14028 was evaluated in a bactericidal assay. The macrophages from resistant cattle were significantly superior (P < 0.05) in controlling intracellular growth of B. abortus, M. bovis BCG, S. dublin but not of S, typhimurium than macrophages from susceptible animals. Controls of all four pathogens correlated strongly with each other in resistant or susceptible macrophages. Data from resistant cattle had a tighter grouping than that of susceptible cattle, while data from susceptible cattle overlapped considerably with data from resistant animals. Therefore, this assay was considered a phenotypic marker of the resistant trait. For each bacterial species a percent bacterial survival value was used as a cut-off point to designate animals as resistant or susceptible. These data were compared with the in vivo challenged resistant or susceptible classification by using the Chi-square analyses. A cut-off point of 70 percent bacterial survival for B. abortus designated 14 cattle as susceptible and seven as resistant and this correlated 100 percent with the number of animals designated as to the relevant category by in vivo challenge. A value of 65 percent bacterial survival for M. bovis BCG, and 100 percent bacterial survival for S. dublin correlated highly with actual numbers of animals designated as susceptible or resistant.