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1.
Food Chem ; 391: 133241, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-35598389

RESUMO

Enterobactin (Ent) is a promising indicator to monitor intestinal level of Enterobacteriaceae for assessment of gut inflammation. In this study, we developed a monoclonal antibody (mAb)-based ELISA for Ent quantification. We immunized mice with an Ent conjugate vaccine. An mAb named 2E4, with the highest anti-Ent antibody titer, was selected for developing indirect competitive ELISA (ic-ELISA). The purified mAb 2E4 showed high affinity (3.1 × 10-10 M) and specificity to Ent. The limit of detection of ic-ELISA was 0.39 µg/mL. The intra- and inter-assay recovery rates of standard curve were up to 94.6% with the coefficients of variation between 4.0% and 12.3%, indicating high accuracy, repeatability, and reproducibility of the ic-ELISA. In addition, the ic-ELISA was able to quantitatively detect Ent produced in different bacterial cultures. Collectively, this study developed an ic-ELISA with excellent performance in Ent quantification, laying a solid foundation for Ent-based diagnostics of gut health.


Assuntos
Enterobactina , Sideróforos , Animais , Anticorpos Monoclonais , Enterobacteriaceae , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos Testes
2.
Sci Rep ; 8(1): 6895, 2018 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720697

RESUMO

Francisella tularensis is a facultative intracellular Gram-negative bacterium that causes the zoonotic disease tularemia. We identified the transcription elongation factor GreA as a virulence factor in our previous study, but its role was not defined. Here, we investigate the effects of the inactivation of the greA gene, generating a greA mutant of F. tularensis subsp. novicida. Inactivation of greA impaired the bacterial invasion into and growth within host cells, and subsequently virulence in mouse infection model. A transcriptomic analysis (RNA-Seq) showed that the loss of GreA caused the differential expression of 196 bacterial genes, 77 of which were identified as virulence factors in previous studies. To confirm that GreA regulates the expression of virulence factors involved in cell invasion by Francisella, FTN_1186 (pepO) and FTN_1551 (ampD) gene mutants were generated. The ampD deletion mutant showed reduced invasiveness into host cells. These results strongly suggest that GreA plays an important role in the pathogenesis of Francisella by affecting the expression of virulence genes and provide new insights into the complex regulation of Francisella infection.


Assuntos
Proteínas de Bactérias/metabolismo , Francisella tularensis/fisiologia , Fatores de Alongamento de Peptídeos/metabolismo , Tularemia/microbiologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biofilmes , Linhagem Celular , Modelos Animais de Doenças , Deleção de Genes , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Estresse Fisiológico , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
3.
Emerg Microbes Infect ; 7(1): 71, 2018 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-29674726

RESUMO

Astroviruses are recognized as a leading cause of gastroenteritis in humans and animals. They are also associated with extra-intestinal diseases, such as hepatitis in ducklings, nephritis in chickens, and encephalitis in cattle. In February 2017, a fatal infection of goslings characterized by visceral urate deposition was reported in the Shandong province, China. Our systematic investigation led to the isolation of an astrovirus, designated AAstV/Goose/CHN/2017/SD01, and similar disease was reproduced by experimental infection of healthy goslings, fulfilling Koch's postulates. The isolated astrovirus replicated well and resulted in 100% mortality of goose embryos. Complete genome sequence analysis revealed that the isolate was genetically distinct from known astroviruses and closely related to members of the avastrovirus genogroup II. Experimental infection showed that the isolate was highly pathogenic in goslings, causing clinical signs, growth repression and in many cases mortality. Histopathological examination indicated that lesions occurred mainly in the kidneys of infected birds. However, virus-specific genomic RNA was detected in all representative tissues, and virus shedding was detected up to 12 days after inoculation, suggesting that the isolate was able to spread systemically and replicate efficiently in vivo. Collectively, our study demonstrates, for the first time, the etiological role of a genetically distinct astrovirus in the fatal infection of goslings.


Assuntos
Infecções por Astroviridae/veterinária , Avastrovirus/genética , Avastrovirus/isolamento & purificação , Gansos/virologia , Gota/veterinária , Doenças das Aves Domésticas/mortalidade , Animais , Animais Domésticos/virologia , Infecções por Astroviridae/epidemiologia , Infecções por Astroviridae/mortalidade , Avastrovirus/classificação , Avastrovirus/patogenicidade , China/epidemiologia , Genoma Viral , Gota/mortalidade , Gota/virologia , Filogenia , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/virologia , Replicação Viral , Eliminação de Partículas Virais , Sequenciamento Completo do Genoma
4.
Emerg Microbes Infect ; 6(7): e66, 2017 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-28745311

RESUMO

Francisella tularensis is a highly infectious intracellular pathogen that infects a wide range of host species and causes fatal pneumonic tularemia in humans. ftlA was identified as a potential virulence determinant of the F. tularensis live vaccine strain (LVS) in our previous transposon screen, but its function remained undefined. Here, we show that an unmarked deletion mutant of ftlA was avirulent in a pneumonia mouse model with a severely impaired capacity to infect host cells. Consistent with its sequence homology with GDSL lipase/esterase family proteins, the FtlA protein displayed lipolytic activity in both E. coli and F. tularensis with a preference for relatively short carbon-chain substrates. FtlA thus represents the first F. tularensis lipase to promote bacterial infection of host cells and in vivo fitness. As a cytoplasmic protein, we found that FtlA was secreted into the extracellular environment as a component of outer membrane vesicles (OMVs). Further confocal microscopy analysis revealed that the FtlA-containing OMVs isolated from F. tularensis LVS attached to the host cell membrane. Finally, the OMV-associated FtlA protein complemented the genetic deficiency of the ΔftlA mutant in terms of host cell infection when OMVs purified from the parent strain were co-incubated with the mutant bacteria. These lines of evidence strongly suggest that the FtlA lipase promotes F. tularensis adhesion and internalization by modifying bacterial and/or host molecule(s) when it is secreted as a component of OMVs.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Francisella tularensis/enzimologia , Francisella tularensis/patogenicidade , Lipase/metabolismo , Macrófagos/microbiologia , Células A549 , Animais , Aderência Bacteriana , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Escherichia coli/metabolismo , Francisella tularensis/genética , Francisella tularensis/fisiologia , Deleção de Genes , Humanos , Fígado/microbiologia , Pulmão/citologia , Camundongos , Mutação , Células RAW 264.7 , Baço/microbiologia , Tularemia/microbiologia , Virulência
5.
Front Microbiol ; 6: 452, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26042101

RESUMO

Upon colonization in the host gastrointestinal tract, the enteric bacterial pathogen Campylobacter jejuni is exposed to a variety of signaling molecules including the catecholamine hormones epinephrine (Epi) and norepinephrine (NE). NE has been observed to stimulate the growth and potentially enhance the pathogenicity of C. jejuni. However, the underlying mechanisms are still largely unknown. In this study, both Epi and NE were also observed to promote C. jejuni growth in MEMα-based iron-restricted medium. Adhesion and invasion of Caco-2 cells by C. jejuni were also enhanced upon exposure to Epi or NE. To further examine the effect of Epi or NE on the pathobiology of C. jejuni, transcriptomic profiles were conducted for C. jejuni NCTC 11168 that was cultured in iron-restricted medium supplemented with Epi or NE. Compared to the genes expressed in the absence of the catecholamine hormones, 183 and 156 genes were differentially expressed in C. jejuni NCTC 11168 that was grown in the presence of Epi and NE, respectively. Of these differentially expressed genes, 102 genes were common for both Epi and NE treatments. The genes differentially expressed by Epi or NE are involved in diverse cellular functions including iron uptake, motility, virulence, oxidative stress response, nitrosative stress tolerance, enzyme metabolism, DNA repair and metabolism and ribosomal protein biosynthesis. The transcriptome analysis indicated that Epi and NE have similar effects on the gene expression of C. jejuni, and provided insights into the delicate interaction between C. jejuni and intestinal stress hormones in the host.

6.
PLoS One ; 10(3): e0115638, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25782009

RESUMO

Taishan Pinus massoniana pollen polysaccharide (TPPPS) improves cellular and humoral immune responses of animals and is a novel potential immunomodulator. However, the components of TPPPS have not been recognized. To investigate the composition of TPPPS, crude polysaccharide was obtained from Taishan P. massoniana pollen through water extraction and ethanol precipitation. Three homogeneous polysaccharide fractions (TPPPS1, TPPPS2, and TPPPS3) were purified from TPPPS by DEAE-cellulose column chromatography. The average molecular weights of the three polysaccharides were 56, 25, and 128 kDa, respectively. Results of high-performance liquid chromatography (HPLC) showed that TPPPS comprised mannose, ribose, xylose, glucuronic acid, galacturonic acid, glucose, galactose, and arabinose. The biological activity assays showed that TPPPS2 and TPPPS3 significantly promoted spleen lymphocyte proliferation, and that TPPPS3 showed better effect than TPPPS2. TPPPS3 enhanced the secretion of cytokine IL-2 and TNF, whereas TPPPS2 mainly elevated IL-2 secretion. By contrast, TPPPS1 exhibited other effects, and it induced the highest amount of NO production, thereby indicating that TPPPS1 had the best antioxidant activity. TPPPS3 at 50 µg/mL significantly inhibited the proliferation of subgroup B Avian Leukosis virus (ALV-B) through virus adsorption interference in vitro. Results indicated that TPPPS comprised three main components, among which, TPPPS1 mainly showed antioxidant effects, whereas TPPPS2 and TPPPS3 played key roles in immunomodulation, especially TPPPS3. Further studies on the use of a reasonable proportion of TPPPS1-3 may facilitate the development of an effective immunomodulator.


Assuntos
Antioxidantes/farmacologia , Antivirais/farmacologia , Fatores Imunológicos/farmacologia , Pinus/química , Pólen/química , Polissacarídeos/farmacologia , Animais , Antioxidantes/química , Antivirais/química , Vírus da Leucose Aviária/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Galinhas , Citocinas/metabolismo , Fatores Imunológicos/química , Peso Molecular , Monossacarídeos/análise , Óxido Nítrico/biossíntese , Polissacarídeos/química , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo
7.
Int Immunopharmacol ; 15(4): 661-5, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23507193

RESUMO

To determine the immune function of Taishan Robinia pseudoacacia Polysaccharide (TRPPS) on chickens, 240 chickens were selected as experimental animals and treated with various doses of TRPPS by hypodermic injection before immunized NDV inactivated vaccine. The results indicated that any dose of TRPPS could significantly promote the development of the immune organs, increase the quantity of leukocyte and the ratio of lymphocyte, and improve the antibody titers against Newcastle disease. Meanwhile, it also increased the magnitude of SIgA in duodenum. However, the dose of 200 mg/ml showed to be the most effective. Therefore, in terms of improving immunologic function and production performance, TRPPS could be used as a vaccine immunopotentiator for immune responses.


Assuntos
Adjuvantes Imunológicos/uso terapêutico , Galinhas/imunologia , Doença de Newcastle/prevenção & controle , Polissacarídeos/uso terapêutico , Doenças das Aves Domésticas/prevenção & controle , Robinia/química , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Duodeno/efeitos dos fármacos , Duodeno/imunologia , Imunidade Humoral/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Imunoglobulina A Secretora/imunologia , Contagem de Leucócitos , Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/imunologia , Polissacarídeos/administração & dosagem , Polissacarídeos/isolamento & purificação , Doenças das Aves Domésticas/imunologia , Vacinas de Produtos Inativados , Vacinas Virais/imunologia
8.
Int J Biol Macromol ; 56: 94-8, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23403027

RESUMO

Three adjuvants, namely, Taishan Pinus massoniana pollen polysaccharide (TPPPS), white mineral oil (WO) and propolis (PP), were added to the outer membrane protein (OMP) of Proteus mirabilis (P. mirabilis) and their effects were compared. Three hundred 1-day-old chicks were randomly divided into five groups (I-V), with 60 chicks per group, and injected subcutaneously with WO-OMP vaccine (I), PP-OMP vaccine (II), TPPPS-OMP vaccine (III), OMP-only vaccine (IV) and physiological saline (V) at 3, 7 and 12 days old. On days 3, 7, 14, 21, 28, 35, 42 and 49 after the first vaccination, the antibody titers, interleukin-2 levels (IL-2) and T-lymphocyte proliferation rates in the peripheral blood as well as the secreting-type IgA levels (SIgA) in the duodenum were measured. On day 7 after the third vaccination, the chicks were challenged with P. mirabilis strain Q1 and the protective effects of each group were observed. The highest protective rate was observed in group III. Moreover, the antibody titers as well as IL-2, SIgA and T-lymphocyte proliferation rates in this group significantly increased and were significantly higher than those in the other groups at most time points. The results indicate that TPPPS could significantly enhance the effects of the subunit vaccine of P. mirabilis; induced stronger humoral, cellular and mucosal immunity as compared with WO and PP; and should be developed as a vaccine adjuvant.


Assuntos
Vacinas Bacterianas/imunologia , Galinhas/imunologia , Galinhas/microbiologia , Polissacarídeos/farmacologia , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antibacterianos/sangue , Proliferação de Células , Galinhas/sangue , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Duodeno/microbiologia , Duodeno/patologia , Eletroforese em Gel de Poliacrilamida , Imunoglobulina A Secretora/metabolismo , Interleucina-2/sangue , Masculino , Proteínas de Membrana/imunologia , Infecções por Proteus/imunologia , Infecções por Proteus/microbiologia , Infecções por Proteus/prevenção & controle , Linfócitos T/citologia , Linfócitos T/imunologia , Vacinação
9.
Microb Pathog ; 54: 40-5, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22990029

RESUMO

Multiple infections of Bordetella avium (B. avium) with virus, especially immunosuppressive virus, have become more and more severe in chickens in China. The increasing morbidity and mortality of its complications have amplified concerns about the impact of B. avium on animal health. To evaluate the pathogenicity of B. avium under immunosuppression status, we developed four types of Reticuloendotheliosis virus (REV) infection models. After a comparison of body weight, relative immune organ index, Newcastle disease virus antibody titers and lymphocyte ratio, we chose the early age with low dose infection as our immunosuppressive model. To investigate the pathogenicity of B. avium under this model, a study was completed in which chickens were inoculated with REV-only, B. avium-only, both agents (REV -B. avium) or first inoculated with REV and 5 d later with B. avium (REV/B. avium). Results revealed that antibody titers to B. avium, concentrations of IFN-γ and SIgA were decreased in coinfected chickens when compared to the B. avium-only chickens, but the changing trend was similar between REV/B. avium and B. avium-only groups. Overall, REV did enhance the pathogenicity of B. avium. However, B. avium-only did not cause severe immune dysfunction unless chicks were coinfected with REV. REV preceding infection with B. avium showed mild impairment, which needs further exploration.


Assuntos
Infecções por Bordetella/patologia , Bordetella avium/patogenicidade , Hospedeiro Imunocomprometido , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/patologia , Infecções por Retroviridae/complicações , Organismos Livres de Patógenos Específicos , Animais , Infecções por Bordetella/imunologia , Infecções por Bordetella/microbiologia , Galinhas , China , Coinfecção/imunologia , Coinfecção/microbiologia , Coinfecção/patologia , Coinfecção/virologia , DNA Bacteriano/química , DNA Bacteriano/genética , Modelos Animais de Doenças , Imunoglobulina A Secretora/sangue , Terapia de Imunossupressão , Interferon gama/sangue , Dados de Sequência Molecular , Vírus da Reticuloendoteliose/imunologia , Vírus da Reticuloendoteliose/patogenicidade , Infecções por Retroviridae/imunologia , Análise de Sequência de DNA
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