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1.
Vet Microbiol ; 174(3-4): 448-455, 2014 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-25457368

RESUMEN

Lawsonia intracellularis is the aetiological agent of the commercially significant porcine disease, proliferative enteropathy. Current understanding of host-pathogen interaction is limited due to the fastidious microaerophilic obligate intracellular nature of the bacterium. In the present study, expression of bacterial proteins during infection was investigated using a mass spectrometry approach. LC-ESI-MS/MS analysis of two isolates of L. intracellularis from heavily-infected epithelial cell cultures and database mining using fully annotated L. intracellularis genome sequences identified 19 proteins. According to the Clusters of Orthologous Groups (COG) functional classification, proteins were identified with roles in cell metabolism, protein synthesis and oxidative stress protection; seven proteins with putative or unknown function were also identified. Detailed bioinformatic analyses of five uncharacterised proteins, which were expressed by both isolates, identified domains and motifs common to other outer membrane-associated proteins with important roles in pathogenesis including adherence and invasion. Analysis of recombinant proteins on Western blots using immune sera from L. intracellularis-infected pigs identified two proteins, LI0841 and LI0902 as antigenic. The detection of five outer membrane proteins expressed during infection, including two antigenic proteins, demonstrates the potential of this approach to interrogate L. intracellularis host-pathogen interactions and identify novel targets which may be exploited in disease control.


Asunto(s)
Infecciones por Desulfovibrionaceae/veterinaria , Genoma Bacteriano/genética , Enfermedades Intestinales/veterinaria , Lawsonia (Bacteria)/metabolismo , Proteómica , Enfermedades de los Porcinos/microbiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Biología Computacional , Infecciones por Desulfovibrionaceae/inmunología , Infecciones por Desulfovibrionaceae/metabolismo , Infecciones por Desulfovibrionaceae/virología , Interacciones Huésped-Patógeno , Enfermedades Intestinales/microbiología , Lawsonia (Bacteria)/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Porcinos , Espectrometría de Masas en Tándem
2.
Ticks Tick Borne Dis ; 5(4): 415-22, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24685441

RESUMEN

Ticks transmit various human and animal microbial pathogens and may harbour more than one pathogen simultaneously. Both viruses and bacteria can trigger, and may subsequently suppress, vertebrate host and arthropod vector anti-microbial responses. Microbial coinfection of ticks could lead to an advantage or disadvantage for one or more of the microorganisms. In this preliminary study, cell lines derived from the ticks Ixodes scapularis and Ixodes ricinus were infected sequentially with 2 arthropod-borne pathogens, Borrelia burgdorferi s.s., Ehrlichia ruminantium, or Semliki Forest virus (SFV), and the effect of coinfection on the replication of these pathogens was measured. Prior infection of tick cell cultures with the spirochaete B. burgdorferi enhanced subsequent replication of the rickettsial pathogen E. ruminantium whereas addition of spirochaetes to cells infected with E. ruminantium had no effect on growth of the latter. Both prior and subsequent presence of B. burgdorferi also had a positive effect on SFV replication. Presence of E. ruminantium or SFV had no measurable effect on B. burgdorferi growth. In tick cells infected first with E. ruminantium and then with SFV, virus replication was significantly higher across all time points measured (24, 48, 72h post infection), while presence of the virus had no detectable effect on bacterial growth. When cells were infected first with SFV and then with E. ruminantium, there was no effect on replication of either pathogen. The results of this preliminary study indicate that interplay does occur between different pathogens during infection of tick cells. Further study is needed to determine if this results from direct pathogen-pathogen interaction or from effects on host cell defences, and to determine if these observations also apply in vivo in ticks. If presence of one pathogen in the tick vector results in increased replication of another, this could have implications for disease transmission and incidence.


Asunto(s)
Infecciones por Alphavirus/transmisión , Borrelia burgdorferi/fisiología , Ehrlichia ruminantium/fisiología , Hidropericardio/transmisión , Ixodes/microbiología , Enfermedad de Lyme/transmisión , Virus de los Bosques Semliki/fisiología , Infecciones por Alphavirus/virología , Animales , Línea Celular , Coinfección , Genes Reporteros , Hidropericardio/microbiología , Humanos , Enfermedad de Lyme/microbiología , Replicación Viral
3.
Ticks Tick Borne Dis ; 3(5-6): 349-54, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23140894

RESUMEN

Tick cell lines play an important role in research on ticks and tick-borne pathogenic and symbiotic microorganisms. In an attempt to derive continuous Dermacentor reticulatus cell lines, embryo-derived primary cell cultures were set up from eggs laid by field ticks originally collected as unfed adults in The Netherlands and maintained for up to 16 months. After several months, it became evident that cells in the primary cultures were infected with a Rickettsia-like intracellular organism. Supernatant medium containing some D. reticulatus cells was inoculated into cultures of 2 Rhipicephalus (Boophilus) microplus cell lines, BME/CTVM2 and BME/CTVM23, where abundant growth of the bacteria occurred intracellularly on transfer to both cell lines. Bacterial growth was monitored by light (live, inverted microscope, Giemsa-stained cytocentrifuge smears) and transmission electron microscopy revealing heavy infection with typical intracytoplasmic Rickettsia-like bacteria, not present in uninfected cultures. DNA was extracted from bacteria-infected and uninfected control cultures, and primers specific for Rickettsia 16S rRNA, ompB, and sca4 genes were used to generate PCR products that were subsequently sequenced. D. reticulatus primary cultures and both infected tick cell lines were positive for all 3 Rickettsia genes. Sequencing of PCR products revealed 99-100% identity with published Rickettsia raoultii sequences. The R. raoultii also grew abundantly in the D. nitens cell line ANE58, poorly in the D. albipictus cell line DALBE3, and not at all in the D. andersoni cell line DAE15. In conclusion, primary tick cell cultures and cell lines are useful systems for isolation and propagation of fastidious tick-borne microorganisms. In vitro isolation of R. raoultii from Dutch D. reticulatus confirms previous PCR-based detection in field ticks, and presence of the bacteria in the tick eggs used to initiate the primary cultures confirms that transovarial transmission of this Rickettsia occurs.


Asunto(s)
Dermacentor/microbiología , Rickettsia/aislamiento & purificación , Animales , Proteínas Bacterianas/genética , Técnicas Bacteriológicas/métodos , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Femenino , Microscopía , Datos de Secuencia Molecular , Países Bajos , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN
4.
Ticks Tick Borne Dis ; 3(3): 137-46, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22743047

RESUMEN

As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed 'tick-only' viruses inhabiting tick cell lines.


Asunto(s)
Vectores Arácnidos/microbiología , Argasidae/microbiología , Bacterias/genética , Ixodidae/microbiología , Virus ARN/genética , Animales , Vectores Arácnidos/virología , Argasidae/ultraestructura , Argasidae/virología , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , ADN Complementario/genética , Humanos , Ixodidae/ultraestructura , Ixodidae/virología , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Virus ARN/ultraestructura , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , ARN Viral/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad de la Especie , Simbiosis , Virión/ultraestructura
5.
Clin Vaccine Immunol ; 18(8): 1282-7, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21697340

RESUMEN

Investigation of antigenic determinants of the microaerophilic obligate intracellular bacterium Lawsonia intracellularis using a mass spectrometry approach identified a novel bacterial protein present in an extract of cell culture medium aspirated from heavily infected in vitro cell cultures. Western immunoblotting analysis of SDS-PAGE-resolved proteins using immune sera pooled from L. intracellularis-infected pigs revealed the presence of a strongly immunoreactive band of ∼ 72 kDa. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of this component and database mining using a fully annotated L. intracellularis genome sequence and the comprehensive GenBank prokaryotic genomic database highlighted the presence of a protein that shares little sequence similarity with other prokaryotic proteins and appears to be highly species specific. Detailed bioinformatic analyses identified the protein as member of the autotransporter protein family of surface-exposed proteins, and the designation LatA (Lawsonia autotransporter protein A) is suggested. Recognition of recombinant LatA on Western blots by a panel of sera from infected and control pigs corresponded 100% with a commercial serodiagnostic that relies on in vitro culture of this fastidious organism. LatA therefore represents a potential candidate for the development of a rapid and species-specific serodiagnostic reagent.


Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Lawsonia (Bacteria)/inmunología , Lawsonia (Bacteria)/metabolismo , Proteínas de Transporte de Membrana/inmunología , Proteínas de Transporte de Membrana/metabolismo , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/análisis , Western Blotting , Biología Computacional , Infecciones por Desulfovibrionaceae/diagnóstico , Infecciones por Desulfovibrionaceae/veterinaria , Inmunoensayo , Lawsonia (Bacteria)/química , Espectrometría de Masas , Proteínas de Transporte de Membrana/análisis , Porcinos
6.
Vet Microbiol ; 139(3-4): 298-303, 2009 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-19589649

RESUMEN

Contact-dependent secretion systems, such as the type III secretion system (T3SS), have been shown to play significant roles in the pathogenicity of many gram-negative bacterial pathogens. Lawsonia intracellularis is a novel, obligate intracellular gram-negative bacterium, which has been identified as the etiological agent of proliferative enteropathies in numerous animal species. Analysis of the genome sequence of the L. intracellularis strain PHE/MN1-00 has revealed the presence of a T3SS secretion system in this bacterium. In this study we aimed to determine whether this important virulence mechanism is also present in L. intracellularis strain LR189/5/83. Using a PCR-based approach, we verified the presence of a genomic region encoding a T3SS. Specifically, a gene highly homologous to the yscN energiser component of the prototypic T3SS of Yersinia spp. was identified and termed lscN. Two further open reading frames (ORFs) contiguous with lscN were also identified: lscO and lscQ, which are also homologues of ORFs within the T3SS of Yersinia spp. To establish whether this T3SS may be functional, expression was monitored directly by RT-PCR and indirectly by detection of serological responses in vaccinated and infected animals. Transcripts for lscN and lscQ were detected and purified rLscQ was recognized by antiserum from infected pigs, indicating expression in vivo during infection. By analogy to other bacteria, this T3SS may be crucial for intracellular development and is likely to play a significant role in the virulence of this unusual pathogen.


Asunto(s)
Lawsonia (Bacteria)/metabolismo , Lawsonia (Bacteria)/patogenicidad , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Lawsonia (Bacteria)/genética , Datos de Secuencia Molecular , ARN Bacteriano/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Porcinos , Virulencia
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