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Pneumonia in cattle is one of the causes of morbidity rates and economic loss. The host response to lung infections caused by Ureaplasma diversum in bovines is virtually unknown. Here in the immune response was evaluated in a murine model for an experimental pulmonary infection by U. diversum. Therefore, AJ, BALB/C and C57BL/6 mice received intratracheal inoculation of U. diversum and were evaluated after 1, 2, 3, 7 and 14 days and the clinical specimens were collected. In bronchoalveolar lavages (BAL) an increase of inflammatory cells was observed. Neutrophils were the main cells recruited to the site of infection and the infiltration was coincided with the production of pro-inflammatory cytokines. We found a large amount of neutrophil in this initial period, followed by a decrease 7 and 14 days post infection, accompanied by bacterial clearance. Our results evidenced the presence of U. diversum within the neutrophil that suggests a phagocytic role of this cell in the elimination of the infection. The immune response features reported here are the initial evidence that healthy immune systems may control these microorganisms. This may be the first step to design new strategies immune based to control the infections in naturally infected hosts.
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Neumonía , Infecciones por Ureaplasma , Animales , Bovinos , Pulmón , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neutrófilos , UreaplasmaRESUMEN
BACKGROUND: Ureaplasma diversum is a pathogen found in the genital tract of cattle and associated with genital disorders such as infertility, placentitis, abortion, birth of weak calves, low sperm motility, seminal vesiculitis and epididymitis. There are few studies evaluating the genetic diversity of U. diversum strains and their influence on the immune response in cattle. Therefore, to better understand genetic relationships of the pathogenicity of U. diversum, a multilocus sequence typing (MLST) scheme was performed to characterize the ATCC 49782 strain and another 40 isolates recovered from different Brazilian states. RESULTS: Primers were designed for housekeeping genes ftsH, polC, rpL22, rpoB, valS and ureA and for virulence genes, phospholipase D (pld), triacylglycerol lipase (tgl), hemolysin (hlyA), MIB-MIP system (mib,mip), MBA (mba), VsA (VsA) and ribose transporter (tABC). PCRs were performed and the targeted gene products were purified and sequenced. Sequence types (STs), and clonal complexes (CCs) were assigned and the phylogenetic relationship was also evaluated. Thus, a total of 19 STs and 4 CCs were studied. Following the molecular analysis, six isolates of U. diversum were selected, inoculated into bovine monocyte/macrophage culture and evaluated for gene expression of the cytokines TNF-α, IL-1, IL-6, IL-10 and IL-17. Differences were detected in the induction of cytokines, especially between isolates 198 and BA78, promoted inflammatory and anti-inflammatory profiles, respectively, and they also differed in virulence factors. CONCLUSION: It was observed that intra-species variability between isolates of U. diversum can induce variations of virulent determinants and, consequently, modulate the expression of the triggered immune response.
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Infecciones por Ureaplasma/veterinaria , Ureaplasma/genética , Ureaplasma/inmunología , Animales , Brasil , Bovinos , Enfermedades de los Bovinos/microbiología , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Femenino , Masculino , Tipificación de Secuencias Multilocus/veterinaria , Filogenia , Ureaplasma/clasificación , Ureaplasma/patogenicidad , Infecciones por Ureaplasma/inmunología , Virulencia/genéticaRESUMEN
Objectives:Ureaplasma diversum is a pathogen of cows that may cause intense inflammatory responses in the reproductive tract and interfere with bovine reproduction. The aims of this study were to evaluate the immune response of bovine blastocysts and macrophages to U. diversum infection and to evaluate the invasion capacity of this microorganism in bovine blastocysts. Methods: Viable and heat-inactivated U. diversum strains ATCC 49782 and CI-GOTA and their extracted membrane lipoproteins were inoculated in macrophages in the presence or absence of signaling blockers of Toll-Like Receptor (TLR) 4, TLR2/4, and Nuclear Factor KB (NF-κB). In addition, the same viable U. diversum strains were used to infect bovine blastocysts. RNA was extracted from infected and lipoprotein-exposed macrophages and infected blastocysts and assayed by qPCR to evaluate the expression of Interleukin 1 beta (IL-1ß), Tumor Necrosis Factor Alpha (TNF-α), TLR2 and TLR4 genes. U. diversum internalization in blastocysts was followed by confocal microscopy. Results: Both Ureaplasma strains and different concentrations of extracted lipoproteins induced a higher gene expression of IL-1ß, TNF-α, TLR2, and TLR4 in macrophages (p < 0.05) when compared to non-infected cells. The used blockers inhibited the expression of IL-1ß and TNF-α in all treatments. Moreover, U. diversum was able to internalize within blastocysts and induce a higher gene expression of IL-1b and TNF- α when compared to non-infected blastocysts (p < 0.05). Conclusion: The obtained results strongly suggest that U. diversum and its lipoproteins interact with TLR4 in a signaling pathway acting via NF-kB signaling to stimulate the inflammatory response. This is the first study to evaluate the in vitro immunological response of macrophages and bovine blastocysts against U. diversum. These results may contribute to a better understanding of the immunomodulatory activity and pathogenicity of this infectious agent.
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OBJECTIVE: To detect Mollicutes in women with endometriosis and healthy peritoneal tissues and evaluate the participation of these bacteria in the immune response during endometriosis. DESIGN: Cross-sectional study. SETTING: University hospitals. PATIENT (S): Women with endometriosis (n = 73) and without endometriosis (n = 31). INTERVENTION(S): Endocervical swabs, peritoneal fluid, and biopsied lesions of endometriosis of women with endometriosis (study group) and healthy peritoneal tissues (control group) were collected during surgery. Clinical characteristics were registered before surgery. MAIN OUTCOME MEASURE(S): We determined the infectious agents with the use of quantitative polymerase chain reaction (PCR). The cytokine secretion profile was determined with the use of Luminex. The expression of immune response related genes was determined with the use of a PCR array kit. RESULT(S): All target microorganisms were detected at least once in the swab samples analyzed. It was possible to observe higher diversity of microorganisms in the samples of swab and peritoneal fluid in the study group compared with the control. Ureaplasma parvum was associated with the severity of the symptom dyspareunia. Mycoplasma genitalium was associated with higher production of interferon-γ and interleukin-1ß. Genes of inflammatory response activation and antigen presentation were up-regulated in biopsied tissue of women with endometriosis. In women with endometriosis, peritoneal fluid cells showed a down-regulation of genes associated with the inflammatory response. This down-regulation profile was higher in presence of M. genitalium. CONCLUSION(S): Mycoplasma genitalium may play a key role in the immune tolerance process and, especially, the aggravation of this profile. More studies are needed to understand this immune tolerance profile of bacterial infections.
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Endometriosis/inmunología , Endometriosis/microbiología , Tolerancia Inmunológica , Infecciones por Mycoplasma/inmunología , Infecciones por Mycoplasma/microbiología , Mycoplasma genitalium/inmunología , Adolescente , Adulto , Líquido Ascítico/inmunología , Líquido Ascítico/microbiología , Estudios de Casos y Controles , Cuello del Útero/inmunología , Cuello del Útero/microbiología , Estudios Transversales , Endometriosis/genética , Endometriosis/metabolismo , Femenino , Regulación de la Expresión Génica , Hospitales Universitarios , Interacciones Huésped-Patógeno , Humanos , Tolerancia Inmunológica/genética , Mediadores de Inflamación/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Persona de Mediana Edad , Infecciones por Mycoplasma/genética , Infecciones por Mycoplasma/metabolismo , Mycoplasma genitalium/patogenicidad , Adulto JovenRESUMEN
Whole genome sequencing and analyses of Ureaplasma diversum ATCC 49782 was undertaken as a step towards understanding U. diversum biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)-Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome (lipoproteins, multiple-banded antigen like protein, membrane nuclease lipoprotein and variable surface antigens lipoprotein). In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of U. diversum was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable U. diversum. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, U. diversum has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis.
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Genoma Bacteriano/genética , Interacciones Huésped-Patógeno/genética , Infecciones por Ureaplasma/genética , Ureaplasma/genética , Composición de Base/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inflamasomas/genética , Lipoproteínas/genética , Redes y Vías Metabólicas/genética , Anotación de Secuencia Molecular , Mycoplasma/genética , Mycoplasma/patogenicidad , Fosfolipasas/genética , Receptores Toll-Like/genética , Ureaplasma/patogenicidad , Infecciones por Ureaplasma/microbiología , Infecciones por Ureaplasma/patología , Ureasa/genéticaRESUMEN
Here, we report the complete genome sequence of Ureaplasma diversum strain ATCC 49782. This species is of bovine origin, having an association with reproductive disorders in cattle, including placentitis, fetal alveolitis, abortion, and birth of weak calves. It has a small circular chromosome of 975,425 bp.