RESUMO
BACKGROUND: Escherichia coli O157:H7 (O157) has been linked to numerous foodborne disease outbreaks. The ability to rapidly sequence and analyze genomes is important for understanding epidemiology, virulence, survival, and evolution of outbreak strains. In the current study, we performed comparative genomics to determine structural and functional features of the genome of a foodborne O157 isolate NADC 6564 and infer its evolutionary relationship to other O157 strains. RESULTS: The chromosome of NADC 6564 contained 5466 kb compared to reference strains Sakai (5498 kb) and EDL933 (5547 kb) and shared 41 of its 43 Linear Conserved Blocks (LCB) with the reference strains. However, 18 of 41 LCB had inverse orientation in NADC 6564 compared to the reference strains. NADC 6564 shared 18 of 19 bacteriophages with reference strains except that the chromosomal positioning of some of the phages differed among these strains. The additional phage (P19) of NADC 6564 was located on a 39-kb insertion element (IE) encoding several hypothetical proteins, an integrase, transposases, transcriptional regulators, an adhesin, and a phosphoethanolamine transferase (PEA). The complete homologs of the 39-kb IE were found in E. coli PCN061 of porcine origin. The IE-encoded PEA showed low homology (32-33%) to four other PEA in NADC 6564 and PEA linked to mobilizable colistin resistance in E. coli but was highly homologous (95%) to a PEA of uropathogenic, avian pathogenic, and enteroaggregative E. coli. NADC 6564 showed slightly higher minimum inhibitory concentration of colistin compared to the reference strains. The 39-kb IE also contained dndBCDE and dptFGH operons encoding DNA S-modification and a restriction pathway, linked to oxidative stress tolerance and self-defense against foreign DNA, respectively. Evolutionary tree analysis grouped NADC 6564 with lineage I O157 strains. CONCLUSIONS: These results indicated that differential phage counts and different chromosomal positioning of many bacteriophages and genomic islands might have resulted in recombination events causing altered chromosomal organization in NADC 6564. Evolutionary analysis grouped NADC 6564 with lineage I strains and suggested its earlier divergence from these strains. The ability to perform S-DNA modification might affect tolerance of NADC 6564 to various stressors.
Assuntos
Escherichia coli O157/genética , Microbiologia de Alimentos , Genômica , Bacteriófagos/fisiologia , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis/genética , Escherichia coli O157/enzimologia , Escherichia coli O157/virologia , Evolução Molecular , Genoma Bacteriano/genética , Ilhas Genômicas/genética , Óperon/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated with Leishmania major and trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated with L. major alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected with L. major alone, coinoculated with carrier beads and L. major, or coinoculated with trimannose-coated beads and L. major Trimannose treatment of L. major-infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR-/-) mice lack the ability to detect trimannose. When MR-/- mice were infected with L. major and treated with trimannose beads, they did not have decreased lesion size. Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis.
Assuntos
Leishmania major , Leishmaniose Cutânea/patologia , Manose/análogos & derivados , Animais , Feminino , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Masculino , Manose/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microesferas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismoRESUMO
Enterohemorrhagic Escherichia coli O157:H7 colonizes the gastrointestinal tract of ruminants, including cattle and bison, which are reservoirs of these zoonotic disease-causing bacteria. Healthy animals colonized by E. coli O157:H7 do not experience clinical symptoms of the disease induced by E. coli O157:H7 infections in humans; however, a variety of host immunological factors may play a role in the amount and frequency of fecal shedding of E. coli O157:H7 by ruminant reservoirs. How gastrointestinal colonization by E. coli O157:H7 impacts these host animal immunological factors is unknown. Here, various isogenic mutant strains of a foodborne isolate of E. coli O157:H7 were used to evaluate bacterial killing capacity of macrophages of cattle and bison, the two ruminant species. Cattle macrophages demonstrated an enhanced ability to phagocytose and kill E. coli O157:H7 compared to bison macrophages, and killing ability was impacted by E. coli O157:H7 virulence gene expression. These findings suggest that the macrophage responses to E. coli O157:H7 might play a role in the variations observed in E. coli O157:H7 fecal shedding by ruminants in nature.
Assuntos
Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/genética , Macrófagos/imunologia , Macrófagos/microbiologia , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/genética , Derrame de Bactérias , Bison/imunologia , Bison/microbiologia , Bovinos/imunologia , Bovinos/microbiologia , Doenças dos Bovinos/microbiologia , Linhagem Celular , Infecções por Escherichia coli/imunologia , Escherichia coli O157/imunologia , Fezes/microbiologia , Doenças Transmitidas por Alimentos/microbiologia , Trato Gastrointestinal/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Fagocitose , Fenótipo , Fosfoproteínas/genética , Ruminantes/microbiologia , Virulência , Zoonoses/microbiologiaRESUMO
During visceral leishmaniasis (VL), Th1-based inflammation is induced to control intracellular parasites. Inflammation-based pathology was shown to be dampened by IL-10 and eventual programmed death 1-mediated T cell exhaustion. Cell type(s) responsible for the initiation of T cell-produced IL-10 during VL are unknown. CD19(+), CD5(-), CD1d(-), IgD(hi) regulatory B cells from healthy controls produced IL-10 in the absence of infection or stimulation, in contrast to IgD(lo/neg) B cells. IgD(hi) B cells may have a de novo versus induced regulatory program. The population of IgD(hi) B cells increased 3-fold as VL progressed. B cells from VL dogs were necessary and sufficient to suppress Th1 cell effector function. IgD(hi) B cells induced IL-10 production by T cells and IgD(lo) B cells. Blockage of B cell-specific PD-L1 restored Th1 responses. IgD(hi) regulatory B cells represent a novel regulatory B cell that may precipitate T cell exhaustion during VL.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos B Reguladores/imunologia , Antígeno B7-H1/metabolismo , Interleucina-10/metabolismo , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Proteínas de Protozoários/imunologia , Células Th1/imunologia , Animais , Anticorpos Bloqueadores/metabolismo , Anticorpos Antiprotozoários/metabolismo , Linfócitos B Reguladores/parasitologia , Antígeno B7-H1/imunologia , Células Cultivadas , Progressão da Doença , Cães , Feminino , Humanos , Tolerância Imunológica , Imunoglobulina D/metabolismo , Masculino , Células Th1/parasitologiaRESUMO
Chronic kidney disease is a major contributor to human and companion animal morbidity and mortality. Renal complications are sequelae of canine and human visceral leishmaniasis (VL). Despite the high incidence of infection-mediated glomerulonephritis, little is known about pathogenesis of VL-associated renal disease. Leishmania infantum-infected dogs are a naturally occurring model of VL-associated glomerulonephritis. Membranoproliferative glomerulonephritis type I [24 of 25 (96%)], with interstitial lymphoplasmacytic nephritis [23 of 25 (92%)], and glomerular and interstitial fibrosis [12 of 25 (48%)] were predominant lesions. An ultrastructural evaluation of glomeruli from animals with VL identified mesangial cell proliferation and interposition. Immunohistochemistry demonstrated significant Leishmania antigen, IgG, and C3b deposition in VL dog glomeruli. Asymptomatic and symptomatic dogs had increased glomerular nucleotide-binding domain leucine-rich repeat-containing-like receptor family, pyrin domain containing 3 and autophagosome-associated microtubule-associated protein 1 light chain 3 associated with glomerular lesion severity. Transcriptional analyses from symptomatic dogs confirmed induction of autophagy and inflammasome genes within glomeruli and tubules. On the basis of temporal VL staging, glomerulonephritis was initiated by IgG and complement deposition. This deposition preceded presence of nucleotide-binding domain leucine-rich repeat-containing-like receptor family, pyrin domain containing 3-associated inflammasomes and increased light chain 3 puncta indicative of autophagosomes in glomeruli from dogs with clinical VL and renal failure. These findings indicate potential roles for inflammasome complexes in glomerular damage during VL and autophagy in ensuing cellular responses.
Assuntos
Autofagia/fisiologia , Proteínas de Transporte/metabolismo , Glomerulonefrite/veterinária , Inflamassomos/metabolismo , Leishmania infantum , Leishmaniose Visceral/veterinária , Animais , Cães , Glomerulonefrite/metabolismo , Glomerulonefrite/parasitologia , Glomérulos Renais/metabolismo , Glomérulos Renais/parasitologia , Glomérulos Renais/patologia , Leishmaniose Visceral/complicações , Leishmaniose Visceral/metabolismoRESUMO
Leishmaniasis is a zoonotic disease caused by predominantly vectorborne Leishmania spp. In the United States, canine visceral leishmaniasis is common among hounds, and L. infantum vertical transmission among hounds has been confirmed. We found that L. infantum from hounds remains infective in sandflies, underscoring the risk for human exposure by vectorborne transmission.
Assuntos
Doenças do Cão/transmissão , Transmissão Vertical de Doenças Infecciosas/veterinária , Leishmania infantum/patogenicidade , Zoonoses/transmissão , Animais , Doenças do Cão/epidemiologia , Cães , Humanos , Leishmaniose/epidemiologia , Leishmaniose/veterinária , Psychodidae/patogenicidade , Estados Unidos/epidemiologia , Carga Viral , Zoonoses/patologiaRESUMO
Lipopolysaccharides (LPS) of Leishmania spp are known to alter innate immune responses. However, the ability of these sugars to specifically alter adaptive T-cell responses is unclear. To study cap sugar-T-cell interactions, pathogen mimics (namely glycodendrimer-coated latex beads with acid-labile linkers) were synthesized. Upon lysosomal acidification, linker breakdown releases glycodendrimers for possible loading on antigen presenting molecules to induce T-cell growth. T-cell proliferation was indeed higher after macrophage exposure to mannobioside or -trioside-containing glycodendrimers than to non-functionalized beads. Yet, blocking phagolysosomal acidification only reduced T-cell proliferation with macrophages exposed to beads with an acid-labile-linker and not to covalently-linked beads. These sugar-modified reagents show that oligosaccharides alone can drive T-cell proliferation by acidification-requiring presentation, most significantly in NKT receptor (CD160)-restricted T cells.
Assuntos
Imunidade Adaptativa , Imunidade Inata , Leishmania/imunologia , Lipopolissacarídeos/imunologia , Linfócitos T/imunologia , Linfócitos T/microbiologia , Ácidos/química , Apresentação de Antígeno , Humanos , Técnicas Imunológicas/métodos , Indicadores e Reagentes/química , Leishmania/química , Leishmaniose/parasitologia , Lipopolissacarídeos/química , Macrófagos/imunologia , Macrófagos/microbiologia , Linfócitos T/citologiaRESUMO
Shiga-toxin producing Escherichia coli O157:H7 (O157)-based vaccines can provide a potential intervention strategy to limit foodborne zoonotic transmission of O157. While the peripheral antibody response to O157 vaccination has been characterized, O157-specific cellular immunity at the rectoanal junction (RAJ), a preferred site for O157 colonization, remains poorly described. Vaccine induced mucosal O157-specific antibodies likely provide some protection, cellular immune responses at the RAJ may also play a role in protection. Distinct lymphoid follicles were increased in the RAJ of vaccinated/challenged animals. Additionally, increased numbers of interferon (IFN)γ-producing cells and γδ + T cells were detected in the follicular region of the RAJ of vaccinated/challenged animals. Likewise, adjuvanted-vaccine formulation is critical in immunogenicity of the O157 parenteral vaccine. Local T cell produced IFNγ may impact epithelial cells, subsequently limiting O157 adherence, which was demonstrated using in vitro attachment assays with bovine epithelial cells. Thus, distinct immune changes induced at the mucosa of vaccinated and challenged animals provide insight of mechanisms associated with limiting O157 fecal shedding. Enhancing mucosal immunity may be critical in the further development of efficacious vaccines for controlling O157 in ruminants and thus limiting O157 transmission to humans.
Assuntos
Vacinas Bacterianas/farmacologia , Infecções por Escherichia coli , Escherichia coli O157/imunologia , Imunidade nas Mucosas/efeitos dos fármacos , Interferon gama/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/imunologia , Bovinos , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Infecções por Escherichia coli/veterinária , Humanos , Masculino , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologiaRESUMO
Bovine brucellosis, cause by infection with Brucella abortus, causes reproductive failure in cattle, has a major economic impact to producers, and as a zoonoses, it is a disease of public health concern. Characterization of the protective immune response against Brucella infection is important to our understanding of disease pathogenesis and for the development of diagnostic assays and vaccines. Most of the knowledge regarding protection against Brucella comes from studies in the murine model, but less is known about the immune responses in cattle. Assessment of antigen-specific T cell frequency and functional phenotype are critical to understand the immune status of the host, characterize mechanisms of protective immunity and immunopathology, and to predict immune protection. The frequency of circulating T cells specific for a particular pathogen is often very low, making analysis of such responses difficult. Our goal was to develop a flow-cytometry based approach to better track Brucella-specific T cell responses. Using peripheral blood mononuclear cells (PMBC) from Brucella abortus strain RB51-vaccinated cattle, we optimized an in vitro stimulation protocol based on a combination of antigen and pan-T cell stimulation. We then assessed RB51-specific T cell responses by concurrently measuring proliferation and cytokine production using flow-cytometry. This methodology enhances the detection of peripheral, Brucella-specific responses in cattle following RB51 vaccination. This protocol is versatile in that it can be modified to fit other in vitro stimulation systems and additional functional or phenotypic parameters can be added for flow cytometric detection and characterization of antigen-specific T cells.
Assuntos
Vacina contra Brucelose/administração & dosagem , Brucella/patogenicidade , Brucelose Bovina/prevenção & controle , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imunogenicidade da Vacina , Ativação Linfocitária/efeitos dos fármacos , Animais , Brucella/imunologia , Vacina contra Brucelose/imunologia , Brucelose Bovina/imunologia , Brucelose Bovina/metabolismo , Brucelose Bovina/microbiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/microbiologia , Bovinos , Células Cultivadas , Feminino , Citometria de Fluxo , Interações Hospedeiro-Patógeno , Imunoensaio , Interferon gama/metabolismo , Fatores de Tempo , VacinaçãoRESUMO
Cattle are the asymptomatic reservoirs of Escherichia coli O157:H7 (O157) that preferentially colonizes the bovine recto-anal junction (RAJ). Understanding the influence of O157 on the diversity of the RAJ microbiota could give insights into its persistence at the RAJ in cattle. Hence, we compared changes in bovine RAJ and fecal microbiota following O157 challenge under experimental conditions. Cattle were either orally challenged (n = 4) with1010 CFU of a streptomycin-resistant O157 strain 86-24, or mock-challenged (n = 4) with phosphate buffered saline. Rectoanal mucosal swab (RAMS) and fecal samples were collected at different time points for analysis. Alpha diversity measures (Chao1 species richness and Shannon diversity index) were found to be significantly different between RAMS and fecal samples but not influenced by O157 challenge. The Firmicutes to Bacteroidetes (F: B) ratio was higher in RAMS samples from O157 colonized animals and this may have influenced the consistent yet decreased O157 colonization at the RAJ. Specific bacterial genera that were present in relative low abundance in fecal and RAMS microbiota did not affect overall microbial diversity but were associated with O157 colonization. Differential abundance analysis (DAA) of genera in samples from O157 shedding cattle indicated significantly higher relative abundance of Paenibacillus and Fusobacterium in RAMS, and Tyzzerella in fecal samples. Mock-challenged cattle showed higher relative abundance of Intestinimonas and Citrobacter in RAMS samples, and Succinivibrio, and Prevotella 1 in fecal samples. These results suggest that O157 challenge exerts transient influence on the intestinal microbial community which in turn might promote O157 colonization in a site-specific manner.
RESUMO
Bovine anaplasmosis is the most prevalent tick-transmitted disease of cattle worldwide and a major obstacle to profitable beef production. Use of chlortetracycline-medicated feed to control active anaplasmosis infections during the vector season has raised concerns about the potential emergence of antimicrobial resistance in bacteria that may pose a risk to human health. Furthermore, the absence of effectiveness data for a commercially available, conditionally licensed anaplasmosis vaccine is a major impediment to implementing anaplasmosis control programs. The primary objective of this study was to develop a single-dose vaccine delivery platform to produce long-lasting protective immunity against anaplasmosis infections. Twelve Holstein steers, aged 11 to 12 wk, were administered a novel 3-stage, single-dose vaccine against Anaplasma marginale, a major surface protein 1a. The vaccine consisted of a soluble vaccine administered subcutaneously (s.c.) for immune priming, a vaccine depot of a biodegradable polyanhydride rod with intermediate slow release of the vaccine for boosting immune response, and an immune-isolated vaccine platform for extended antigen release (VPEAR implant) deposited s.c. in the ear. Six calves were randomly assigned to 2 vaccine constructs (nâ =â 3) that featured rods and implants containing a combination of 2 different adjuvants, diethylaminoethyl (DEAE)-Dextran and Quil-A (Group A). The remaining 6 calves were randomly assigned to 2 vaccine constructs (nâ =â 3) that featured rods and implants containing the same adjuvant (either DEAE-Dextran or Quil A) (Group B). Twenty-one months post-implantation, calves were challenged intravenously with A. marginale stabilate and were monitored weekly for signs of fever, decreased packed cell volume (PCV) and bacteremia. Data were analyzed using a mixed-effects model and chi-squared tests (SAS v9.04.01, SAS Institute, Cary, NC). Calves in Group A had higher PCV than calves in Group B (Pâ =â 0.006) at day 35 post-infection. Calves in Group A were less likely to require antibiotic intervention compared with calves in Group B (Pâ =â 0.014). Results indicate that calves exhibited diminished clinical signs of anaplasmosis when antigen was delivered with a combination of adjuvants as opposed to a single adjuvant. This demonstrates the feasibility of providing long-lasting protection against clinical bovine anaplasmosis infections using a subcutaneous ear implant vaccine construct.
Assuntos
Anaplasma marginale , Anaplasmose/prevenção & controle , Vacinas Bacterianas/administração & dosagem , Doenças dos Bovinos/prevenção & controle , Anaplasmose/imunologia , Anaplasmose/microbiologia , Animais , Vacinas Bacterianas/imunologia , Bovinos , Preparações de Ação Retardada , Implantes de Medicamento , MasculinoRESUMO
Shiga toxin-producing Escherichia coli O157:H7 (O157) can cause mild to severe gastrointestinal disease in humans. Cattle are the primary reservoir for O157, which colonizes the intestinal tract without inducing any overt clinical symptoms. Parenteral vaccination can reduce O157 shedding in cattle after challenge and limit zoonotic transmission to humans, although the impact of vaccination and vaccine formulation on cellular and mucosal immune responses are undetermined. To better characterize the cattle immune response to O157 vaccination, cattle were vaccinated with either water-in-oil-adjuvanted, formalin-inactivated hha deletion mutant of Shiga toxin 2 negative (stx2-) O157 (Adj-Vac); non-adjuvanted (NoAdj-Vac); or non-vaccinated (NoAdj-NoVac) and peripheral T cell and mucosal antibody responses assessed. Cattle in Adj-Vac group had a higher percentage of O157-specific IFNγ producing CD4+ and γδ+ T cells in recall assays compared to the NoAdj-Vac group. Furthermore, O157-specific IgA levels detected in feces of the Adj-Vac group were significantly lower in NoAdj-Vac group. Extracts prepared only from Adj-Vac group feces blocked O157 adherence to epithelial cells. Taken together, these data suggest parenteral administration of adjuvanted, inactivated whole-cell vaccines for O157 can induce O157-specific cellular and mucosal immune responses that may be an important consideration for a successful vaccination scheme.
Assuntos
Doenças dos Bovinos/imunologia , Infecções por Escherichia coli/veterinária , Vacinas contra Escherichia coli/imunologia , Vacinação/veterinária , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/imunologia , Derrame de Bactérias , Bovinos , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/prevenção & controle , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Escherichia coli O157 , Proteínas de Escherichia coli/imunologia , Imunidade Celular , Imunidade nas Mucosas , Masculino , Vacinas de Produtos Inativados/imunologiaRESUMO
Vaccination-induced Escherichia coli O157:H7-specific immune responses have been shown to reduce E. coli O157:H7 shedding in cattle. Although E. coli O157:H7 colonization is correlated with perturbations in intestinal microbial diversity, it is not yet known whether vaccination against E. coli O157:H7 could cause shifts in bovine intestinal microbiota. To understand the impact of E. coli O157:H7 vaccination and colonization on intestinal microbial diversity, cattle were vaccinated with two doses of different E. coli O157:H7 vaccine formulations. Six weeks post-vaccination, the two vaccinated groups (Vx-Ch) and one non-vaccinated group (NonVx-Ch) were orally challenged with E. coli O157:H7. Another group was neither vaccinated nor challenged (NonVx-NonCh). Fecal microbiota analysis over a 30-day period indicated a significant (FDR corrected, p <0.05) association of bacterial community structure with vaccination until E. coli O157:H7 challenge. Shannon diversity index and species richness were significantly lower in vaccinated compared to non-vaccinated groups after E. coli O157:H7 challenge (p < 0.05). The Firmicutes:Bacteroidetes ratio (p > 0.05) was not associated with vaccination but the relative abundance of Proteobacteria was significantly lower (p < 0.05) in vaccinated calves after E. coli O157:H7 challenge. Similarly, Vx-Ch calves had higher relative abundance of Paeniclostridium spp. and Christenellaceae R7 group while Campylobacter spp., and Sutterella spp. were more abundant in NonVx-Ch group post-E. coli O157:H7 challenge. Only Vx-Ch calves had significantly higher (p < 0.001) E. coli O157:H7-specific serum IgG but no detectable E. coli O157:H7-specific IgA. However, E. coli O157:H7-specific IL-10-producing T cells were detected in vaccinated animals prior to challenge, but IFN-γ-producing T cells were not detected. Neither E. coli O157:H7-specific IgG nor IgA were detected in blood or feces, respectively, of NonVx-Ch and NonVx-NonCh groups prior to or post vaccinations. Both Vx-Ch and NonVx-Ch animals shed detectable levels of challenge strain during the course of the study. Despite the lack of protection with the vaccine formulations there were detectable shifts in the microbiota of vaccinated animals before and after challenge with E. coli O157:H7.
Assuntos
Escherichia coli O157/imunologia , Escherichia coli O157/fisiologia , Vacinas contra Escherichia coli/imunologia , Microbioma Gastrointestinal/imunologia , Animais , Bovinos , Imunidade Celular/imunologia , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Interferon gama/biossíntese , Fenótipo , Especificidade da Espécie , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/microbiologiaRESUMO
Brucellosis is a bacterial zoonosis and a significant source of economic loss and a major public health concern, worldwide. Bovine brucellosis, as caused primarily by Brucella abortus, is an important cause of reproductive loss in cattle. Vaccination has been the most effective way to reduce disease prevalence contributing to the success of control and eradication programs. Currently, there are no human vaccines available, and despite the success of commercial vaccines for livestock, such as B. abortus strain RB51 (RB51), there is need for development of novel and safer vaccines against brucellosis. In the current study, we report the fabrication of and immune responses to an implantable single dose polyanhydride-based, methanol-killed RB51 antigen containing delivery platform (VPEAR) in cattle. In contrast to animals vaccinated with RB51, we did not observe measurable RB51-specific IFN-γ or IgG responses in the peripheral blood, following initial vaccination with VPEAR. However, following a subsequent booster vaccination with RB51, we observed an anamnestic response in both vaccination treatments (VPEAR and live RB51). The magnitude and kinetics of CD4+ IFN-γ-mediated responses and circulating memory T cell subpopulations were comparable between the two vaccination treatments. Additionally, IgG titers were significantly increased in animals vaccinated with VPEAR as compared to live RB51- vaccinated animals. These data demonstrate that killed antigen may be utilized to generate and sustain memory, IFN-γ-mediated, CD4+ T cell and humoral responses against Brucella in a natural host. To our knowledge, this novel approach to vaccination against intracellular bacteria, such as Brucella, has not been reported before.
RESUMO
Escherichia coli O157:H7 (O157) can cause from a mild diarrheal illness to hemorrhagic colitis and hemolytic uremic syndrome in humans. Cattle are the primary reservoir for O157 and fecal shedding of O157 by these animals is a major risk factor in contamination of cattle hides and carcasses at slaughter. Vaccination is an important strategy to reduce fecal shedding of O157 in cattle. In this study, we evaluated the immunogenicity and efficacy of an inactivated vaccine strain of O157 formulated with an adjuvant. This vaccine strain was deleted of the hha gene enabling high level expression of the locus of enterocyte effacement (LEE) encoded proteins required for O157 colonization in cattle. The inactivated vaccine strain emulsified with the adjuvant or suspended in the phosphate-buffered saline (PBS) was injected in the neck muscles of two groups of weaned calves followed by a booster three weeks later with the corresponding formulation. Animals in groups 3 and 4 were injected similarly with the adjuvant and PBS, respectively. All animals were orally inoculated three weeks post-booster vaccination with a live culture of O157. The animals vaccinated with the adjuvanted vaccine showed higher serum antibody titers to the vaccine strain and shed O157 for a shorter duration and at lower numbers compared to the animals vaccinated with the non-adjuvanted vaccine, adjuvant-only, or PBS. Western blotting of the vaccine strain lysates showed higher immunoreactivity of serum IgG in vaccinated animals to several O157-specific proteins and lipopolysaccharides (LPS). The vaccination induced IgG showed specificity to LEE-encoded proteins and outer membrane LPS as LEE and waaL deletion mutants, unable to produce LEE proteins and synthesize high molecular weight LPS, respectively, yielded significantly lower antibody titers compared to the parent vaccine strain. The positive reactivity of the immune serum was also observed for purified LEE-encoded proteins EspA and EspB. In conclusion, the results of this animal study showed that a two-dose regimen of an adjuvanted vaccine is capable of inducing O157-specific immune response that directly or indirectly reduced fecal shedding of O157 in cattle.
Assuntos
Anticorpos Antibacterianos/sangue , Derrame de Bactérias , Infecções por Escherichia coli/veterinária , Escherichia coli O157/genética , Vacinas contra Escherichia coli/imunologia , Fezes/microbiologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Enterócitos/metabolismo , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Escherichia coli O157/imunologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Vacinas contra Escherichia coli/administração & dosagem , Imunização Secundária , Imunogenicidade da Vacina , Imunoglobulina G/sangue , Mutação , Vacinação/métodos , DesmameRESUMO
Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.
Assuntos
Linfonodos/patologia , RNA/metabolismo , Animais , Animais Selvagens , Bison , BovinosRESUMO
The inflammasome serves as a mechanism by which the body senses damage or danger. These multiprotein complexes form in the cytosol of myeloid, epithelial and potentially other cell types to drive caspase-1 cleavage and the secretion of the pro-inflammatory cytokines IL-1ß and IL-18. Different types of inflammasomes, centered on (and named after) their cytosolic NLRs, respond to signals from bacteria, fungi, and viruses, as well as "sterile inflammatory" triggers. Despite the large body of research accumulated on rodent and human inflammasomes over the past 15 years, only recently have studies expanded to consider the role of inflammasomes in veterinary and wildlife species. Due to the key role of inflammasomes in mediating inflammatory responses observed in humans and rodents, characterization of the similarities and differences between humans/rodents and veterinary species is required to identify genetic and evolutionary influences on disease responses and to develop therapeutic candidates for use in veterinary inflammatory syndromes. Here, we summarize recent findings on inflammasomes in swine, cattle, dogs, bats, small ruminants, and birds. We describe current gaps in our knowledge and highlight promising areas for future research.
Assuntos
Animais Selvagens/imunologia , Bactérias/imunologia , Infecções Bacterianas/veterinária , Interações Hospedeiro-Patógeno/imunologia , Inflamassomos , Gado/imunologia , Animais , Bactérias/patogenicidade , Infecções Bacterianas/imunologia , Caspase 1/imunologia , Bovinos/imunologia , Quirópteros/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Cães/imunologia , Humanos , Inflamação , Interleucina-1beta/imunologia , Ruminantes/imunologia , Transdução de Sinais/imunologia , Suínos/imunologiaRESUMO
Traditionally, vaccination strategies require an initial priming vaccination followed by an antigen boost to generate adequate immunity. Here we describe vaccination against a self-peptide for reproductive sterilization utilizing a three-stage vaccine platform consisting of gonadotropin releasing hormone multiple antigenic peptide (GnRH-MAP) as a soluble injection coupled with subcutaneous administration of polyanhydride-immobilized GnRH-MAP and a cyto-exclusive implant containing GnRH-MAP dendrimer-loaded polyanhydride. This strategy generated and maintained cell-mediated and humoral immunity for up to 41â¯weeks after a single vaccination in mice with enhanced antibody avidity over time. All intact implants had a grossly visible tissue interface with neovascularization and lymphocytic aggregates. Despite detectable immunity, sterility was not achieved and the immune response did not lead to azoospermia in male mice nor prevent estrus and ovulation in female mice. However, the vaccine delivery device is tunable and the immunogen, adjuvants and release rates can all be modified to enhance immunity. This technology has broad implications for the development of long-term vaccination schemes.
Assuntos
Anticorpos/imunologia , Hormônio Liberador de Gonadotropina/imunologia , Polianidridos , Vacinas/administração & dosagem , Vacinas/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos/sangue , Antígenos/química , Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Imunização , Memória Imunológica , Masculino , Camundongos , Polianidridos/química , Vacinas/químicaRESUMO
Tritrichomonas foetus is a flagellated protozoan parasite that causes inflammation of the reproductive tract leading to early embryonic death and abortion in cattle, thereby resulting in significant economic losses. Testing and culling infected bulls is an important strategy for parasite control. Routine testing is mainly limited to bulls that are traveling across state lines or within states that have specific control programs. Both culture and PCR detection methods are available, but they are not typically conducted as part of a yearly breeding soundness program and are not easily conducted in the field. In the present study, we developed a bead agglutination assay for detection of T. foetus antigens. Our experiments revealed that latex beads conjugated to T. foetus lipophosphoglycan-binding antibodies visibly clump in the presence of T. foetus. The detection limit of the assay, determined using both field and laboratory isolates of the parasite, was 0.25µg/mL and 1.0µg/mL total T. foetus antigen, respectively. Our results indicate that an antigen detection test could offer a tool for screening bulls under field conditions.
Assuntos
Antígenos de Protozoários/imunologia , Doenças dos Bovinos/diagnóstico , Infecções Protozoárias em Animais/diagnóstico , Tritrichomonas foetus/isolamento & purificação , Testes de Aglutinação/métodos , Testes de Aglutinação/veterinária , Animais , Anticorpos Monoclonais , Anticorpos Antiprotozoários , Bovinos , Doenças dos Bovinos/parasitologia , Masculino , Reação em Cadeia da Polimerase/veterinária , Infecções Protozoárias em Animais/parasitologiaRESUMO
Leishmania is the causative agent of leishmaniasis, a deadly protozoan disease which affects over 1 million people each year. Autochthonous cases of canine leishmaniasis are generally associated with tropical and subtropical climatic zones. However, in 1999, U.S. hunting dogs were found to have leishmaniasis with no history of travel outside the country. Transmission of this disease was found to be primarily vertical. In endemic areas, dogs are a dominant domestic reservoir host for Leishmania infantum. This study evaluated L. infantum infection prevalence and incidence within US dogs tested over a nine-year span (2007-2015). This investigation used both passive and active surveillance, following an initial outbreak investigation by the Centers for Disease Control. L. infantum infection incidence and prevalence over time and across regions were examined to evaluate whether transmission was sufficient to maintain ongoing infection within this population. These studies also established whether this disease is becoming more or less prominent within this reservoir host, dogs. There was no significant difference between prevalence and incidence rates via as measured by passive vs. active surveillance. Although due to fluctuations in sample submission there were significant changes in both incidence and prevalence of L. infantum in US hunting dogs over this nine year span, these differences were not outside of the interquartile range and therefore there is likely to be a steady-state of transmission within U.S. dogs. Based on these findings, if vertical transmission is the primary means of L. infantum spread in U.S. dogs, with appropriate husbandry and infection control procedures, elimination of L. infantum from US dogs could be possible.