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Combined Transcriptome Sequencing of Mycoplasma hyopneumoniae and Infected Pig Lung Tissue Reveals Up-Regulation of Bacterial F1-Like ATPase and Down-Regulation of the P102 Cilium Adhesin in vivo
Kamminga, Tjerko ; Benis, Nirupama ; Martins dos Santos, Vitor ; Bijlsma, Jetta J.E. ; Schaap, Peter J. - \ 2020
Frontiers in Microbiology 11 (2020). - ISSN 1664-302X
F1-like ATPase - host-pathogen interaction - infection - Mycoplasma hyopneumoniae - P102 cilium adhesin - pathogen enrichment - RNA sequencing
Mycoplasma hyopneumoniae (M. hyopneumoniae) causes enzootic pneumonia in pigs but it is still largely unknown which host-pathogen interactions enable persistent infection and cause disease. In this study, we analyzed the host and bacterial transcriptomes during infection using RNA sequencing. Comparison of the transcriptome of lung lesion tissue from infected pigs with lung tissue from non-infected animals, identified 424 differentially expressed genes (FDR < 0.01 and fold change > 1.5LOG2). These genes were part of the following major pathways of the immune system: interleukin signaling (type 4, 10, 13, and 18), regulation of Toll-like receptors by endogenous ligand and activation of C3 and C5 in the complement system. Besides analyzing the lung transcriptome, a sampling protocol was developed to obtain enough bacterial mRNA from infected lung tissue for RNA sequencing. This was done by flushing infected lobes in the lung, and subsequently enriching for bacterial RNA. On average, 2.2 million bacterial reads were obtained per biological replicate to analyze the bacterial in vivo transcriptome. We compared the in vivo bacterial transcriptome with the transcriptome of bacteria grown in vitro and identified 22 up-regulated and 30 down-regulated genes (FDR < 0.01 and fold change > 2LOG2). Six out of seven genes in the operon encoding the mycoplasma specific F1-like ATPase (MHP_RS02445-MHP_RS02475) and all genes in the operon MHP_RS01965-MHP_RS01990 with functions related to nucleotide metabolism, spermidine transport and glycerol-3-phoshate transport were up-regulated in vivo. Down-regulated in vivo were genes related to glycerol uptake, cilium adhesion (P102), cell division and myo-inositol metabolism. In addition to providing a novel method to isolate bacterial mRNA from infected lung, this study provided insights into changes in gene expression during infection, which could help development of novel treatment strategies against enzootic pneumonia caused by M. hyopneumoniae.
Identification of conditionally essential genes for Streptococcus suis infection in pigs
Arenas, Jesús ; Zomer, Aldert ; Harders-Westerveen, Jose ; Bootsma, Hester J. ; Jonge, Marien I. De; Stockhofe-Zurwieden, Norbert ; Smith, Hilde E. ; Greeff, Astrid De - \ 2020
Virulence 11 (2020)1. - ISSN 2150-5594 - p. 446 - 464.
infection - pathogenesis - Streptococcus suis - Tn-Seq - transposon mutagenesis - zoonotic pathogen
Streptococcus suis is a Gram-positive bacterium and zoonotic pathogen that causes meningitis and sepsis in pigs and humans. The aim of this study was to identify genes required for S. suis infection. We created Tn-Seq libraries in a virulent S. suis strain 10, which was used to inoculate pigs in an intrathecal experimental infection. Comparative analysis of the relative abundance of mutants recovered from different sites of infection (blood, cerebrospinal fluid, and meninges of the brain) identified 361 conditionally essential genes, i.e. required for infection, which is about 18% of the genome. The conditionally essential genes were primarily involved in metabolic and transport processes, regulation, ribosomal structure and biogenesis, transcription, and cell wall membrane and envelope biogenesis, stress defenses, and immune evasion. Directed mutants were created in a set of 10 genes of different genetic ontologies and their role was determined in ex vivo models. Mutants showed different levels of sensitivity to survival in whole blood, serum, cerebrospinal fluid, thermic shock, and stress conditions, as compared to the wild type. Additionally, the role of three selected mutants was validated in co-infection experiments in which pigs were infected with both wild type and isogenic mutant strains. The genetic determinants of infection identified in this work contribute to novel insights in S. suis pathogenesis and could serve as targets for novel vaccines or antimicrobial drugs.
Akkermansia muciniphila reduces Porphyromonas gingivalis-induced inflammation and periodontal bone destruction
Huck, Olivier ; Mulhall, Hannah ; Rubin, George ; Kizelnik, Zev ; Iyer, Radha ; Perpich, John D. ; Haque, Nasreen ; Cani, Patrice D. ; Vos, Willem M. de; Amar, Salomon - \ 2020
Journal of Clinical Periodontology 47 (2020)2. - ISSN 0303-6979 - p. 202 - 212.
infection - inflammation - periodontitis - probiotic
Aim: Akkermansia muciniphila is a beneficial gut commensal, whose anti-inflammatory properties have recently been demonstrated. This study aimed to evaluate the effect of A. muciniphila on Porphyromonas gingivalis elicited inflammation. Material and Methods: In lean and obese mice, A. muciniphila was administered in P. gingivalis-induced calvarial abscess and in experimental periodontitis model (EIP). Bone destruction and inflammation were evaluated by histomorphometric analysis. In vitro, A. muciniphila was co-cultured with P. gingivalis, growth and virulence factor expression was evaluated. Bone marrow macrophages (BMMϕ) and gingival epithelial cells (TIGK) were exposed to both bacterial strains, and the expression of inflammatory mediators, as well as tight junction markers, was analysed. Results: In a model of calvarial infection, A. muciniphila decreased inflammatory cell infiltration and bone destruction. In EIP, treatment with A. muciniphila resulted in a decreased alveolar bone loss. In vitro, the addition of A. muciniphila to P. gingivalis-infected BMMϕ increased anti-inflammatory IL-10 and decreased IL-12. Additionally, A. muciniphila exposure increases the expression of junctional integrity markers such as integrin-β1, E-cadherin and ZO-1 in TIGK cells. A. muciniphila co-culture with P. gingivalis reduced gingipains mRNA expression. Discussion: This study demonstrated the protective effects of A. muciniphila administration and may open consideration to its use as an adjunctive therapeutic agent to periodontal treatment.
Fusarium spp. in Loggerhead Sea Turtles (Caretta caretta): From Colonization to Infection
Cafarchia, Claudia ; Paradies, Romina ; Figueredo, Luciana A. ; Iatta, Roberta ; Desantis, Salvatore ; Bello, Antonio Vito Francesco Di; Zizzo, Nicola ; Diepeningen, Anne D. van - \ 2020
Veterinary Pathology 57 (2020)1. - ISSN 0300-9858 - p. 139 - 146.
bycatch - Caretta caretta - conservation - Fusarium - infection - loggerhead sea turtles - rehabilitation - skin
With the aim of evaluating the presence of Fusarium spp. in sea turtles with and without lesions and assessing the risk factors favoring colonization and/or infection, 74 loggerhead sea turtles (Caretta caretta) admitted to rescue and rehabilitation clinics in Italy were analyzed. The study compared 31 individuals with no apparent macroscopic lesions and 43 individuals with macroscopic lesions. Shell and skin samples were analyzed using Calcofluor white with 10% potassium hydroxide, standard histopathological examination, and fungal cultures. Fusarium spp. were isolated more frequently from animals with superficial lesions (39%) than from those with no macroscopic lesions (16%). Isolates from animals with superficial lesions were Fusarium solani species complex (FSSC) lineages haplotypes 9, 12, and 27 (unnamed lineages), FSSC-2 (Fusarium keratoplasticum), Fusarium oxysporum (27%), and Fusarium brachygibbosum (3%). In contrast, only F. solani haplotypes 9 and 12 were isolated from animals with no macroscopic lesions. The presence of lesions was identified as a risk factor for the occurrence of Fusarium spp. Of the 74 animals, only 7 (9.5%) scored positive on microscopic examination with Calcofluor, and histological examination of those 7 animals revealed necrosis, inflammatory cells, and fungal hyphae in the carapace and skin. The results of this study suggest that fusariosis should be included in the differential diagnosis of shell and skin lesions in sea turtles. Direct examination using Calcofluor and potassium hydroxide was not useful to diagnose the infection. Histopathological examination and fungal culture should be performed to ensure correct treatment and infection control.
In vivo transcriptomes of Streptococcus suis reveal genes required for niche-specific adaptation and pathogenesis
Arenas, Jesús ; Bossers-de Vries, Ruth ; Harders-Westerveen, José ; Buys, Herma ; Ruuls-van Stalle, Lisette M.F. ; Stockhofe-Zurwieden, Norbert ; Zaccaria, Edoardo ; Tommassen, Jan ; Wells, Jerry M. ; Smith, Hilde E. ; Greeff, Astrid de - \ 2019
Virulence 10 (2019)1. - ISSN 2150-5594 - p. 334 - 351.
infection - infectomics - pathogenesis - transcriptomics - zoonotic pathogen
Streptococcus suis is a Gram-positive bacterium and a zoonotic pathogen residing in the nasopharynx or the gastrointestinal tract of pigs with a potential of causing life-threatening invasive disease. It is endemic in the porcine production industry worldwide, and it is also an emerging human pathogen. After invasion, the pathogen adapts to cause bacteremia and disseminates to different organs including the brain. To gain insights in this process, we infected piglets with a highly virulent strain of S. suis, and bacterial transcriptomes were obtained from blood and different organs (brain, joints, and heart) when animals had severe clinical symptoms of infection. Microarrays were used to determine the genome-wide transcriptional profile at different infection sites and during growth in standard growth medium in vitro. We observed differential expression of around 30% of the Open Reading Frames (ORFs) and infection-site specific patterns of gene expression. Genes with major changes in expression were involved in transcriptional regulation, metabolism, nutrient acquisition, stress defenses, and virulence, amongst others, and results were confirmed for a subset of selected genes using RT-qPCR. Mutants were generated in two selected genes, and the encoded proteins, i.e., NADH oxidase and MetQ, were shown to be important virulence factors in coinfection experiments and in vitro assays. The knowledge derived from this study regarding S. suis gene expression in vivo and identification of virulence factors is important for the development of novel diagnostic and therapeutic strategies to control S. suis disease.
Pooling of genital swabs for detection by PCR of Taylorella equigenitalis, the cause of contagious equine metritis
Mawhinney, I. ; Errington, J. ; Stamper, N. ; Torrens, N. ; Engelsma, M.Y. ; Roest, H.I.J. - \ 2019
Equine Veterinary Journal 51 (2019)2. - ISSN 0425-1644 - p. 227 - 230.
diagnosis - horse - infection - validation
Background: Sets of genital swabs are routinely taken from horses to screen for the presence of Taylorella equigenitalis, the cause of contagious equine metritis. Typically, two to four different sites are swabbed at a time and tested by culture or PCR. Objectives: This study explored the feasibility of pooling these swabs for a single PCR test per animal instead of testing each swab individually. Study design: In vitro. Methods: PCR signal strengths (Ct values) from 149 historical PCR positive genital swabs, together with historical data on the number of swabs in a set expected to be positive, were used to assess the suitability of pooling for screening horses for T. equigenitalis infection in the population at large. Twenty-four sets of four equine genital swabs were tested. The sets were prepared in the laboratory using one or more swabs positive for T. equigenitalis from naturally infected cases. Positive and negative swabs were selected to reflect a typical range of PCR Ct values expected in field cases of T. equigenitalis infection. These pools were tested by an established PCR to assess the impact and suitability of a PCR test on pooled swabs compared to individual swab testing, by comparing the Ct values. Results: Pooling one positive swab with three negative swabs produced a small drop in Ct value but all pools were still clearly positive. Main limitations: Large numbers of field positive horses are not available, but the proof of concept approach with laboratory prepared pools shows the method is applicable to field cases. Conclusions: It was concluded that pooling of swabs would confer no appreciable drop in the ability to detect a positive animal compared to individual swab testing; pooling is therefore a suitable alternative to individual swab testing with reduced costs. The Summary is available in Spanish – see Supporting Information.
Quantitative analysis of the dose–response of white spot syndrome virus in shrimp
Ngo, Thuy T.N. ; Senior, Alistair M. ; Culina, Antica ; Santos, Eduardo S.A. ; Vlak, Just M. ; Zwart, Mark P. - \ 2018
Journal of Fish Diseases 41 (2018)11. - ISSN 0140-7775 - p. 1733 - 1744.
dose–response - infection - meta-analysis - modelling - shrimp - white spot syndrome virus
White spot syndrome virus (WSSV) is an important cause of mortality and economic losses in shrimp farming. Although WSSV-induced mortality is virus dose dependent and WSSV infection does not necessarily lead to mortality, the relationships between virus-particle dose, infection and mortality have not been analysed quantitatively. Here, we explored WSSV dose–response by a combination of experiments, modelling and meta-analysis. We performed dose–response experiments in Penaeus vannamei postlarvae, recorded host mortality and detected WSSV infection. When we fitted infection models to these data, two models—differing in whether they incorporated heterogeneous host susceptibility to the virus or not—were supported for two independent experiments. To determine the generality of these results, we reanalysed published data sets and then performed a meta-analysis. We found that WSSV dose–response kinetics is indeed variable over experiments. We could not clearly identify which specific infection model has the most support by meta-analysis, but we argue that these results also are most concordant with a model incorporating varying levels of heterogeneous host susceptibility to WSSV. We have identified suitable models for analysing WSSV dose–response, which can elucidate the most basic virus–host interactions and help to avoid underestimating WSSV infection at low virus doses.
RNA ‘Information Warfare’ in Pathogenic and Mutualistic Interactions
Chaloner, Thomas ; Kan, Jan A.L. van; Grant-Downton, Robert T. - \ 2016
Trends in Plant Science 21 (2016)9. - ISSN 1360-1385 - p. 738 - 748.
fungus - infection - non-coding RNA - pathogen - resistance
Regulatory non-coding RNAs are emerging as key players in host–pathogen interactions. Small RNAs such as microRNAs are implicated in regulating plant transcripts involved in immunity and defence. Surprisingly, RNAs with silencing properties can be translocated from plant hosts to various invading pathogens and pests. Small RNAs are now confirmed virulence factors, with the first report of fungal RNAs that travel to host cells and hijack post-transcriptional regulatory machinery to suppress host defence. Here, we argue that trans-organism movement of RNAs represents a common mechanism of control in diverse interactions between plants and other eukaryotes. We suggest that extracellular vesicles are the key to such RNA movement events. Plant pathosystems serve as excellent experimental models to dissect RNA ‘information warfare’ and other RNA-mediated interactions.
Discovery, characterization and applications of natural DNA transformation in Streptococcus suis
Zaccaria, E. - \ 2015
Wageningen University. Promotor(en): Jerry Wells, co-promotor(en): Peter van Baarlen. - Wageningen : Wageningen University - ISBN 9789462576056 - 171
streptococcus suis - virulence - pathogenesis - gene expression - direct dna uptake - virulence factors - infection - modeling - streptococcus suis - virulentie - pathogenese - genexpressie - directe dna-opname - virulente factoren - infectie - modelleren
Streptococcus suis is Gram-positive bacterium and its natural habitat is the upper respiratory tract of pigs, and in particular the tonsils and nasal cavity. Although it is considered to be a normal member of the adult pig microbiome, it can cause serious diseases in pigs and humans. S. suis is in fact one of the most important swine pathogens world-wide, causing a wide variety of diseases in pigs including septicemia, arthritis, endocarditis, and meningitis that leads often to a rapid death within 1-2 days. Although most human infections are considered the consequence of occupational exposure, in the last years the number of human cases has increased and isolates with multi-resistance genes have been isolated. Human infection caused by S. suis are characterized by a similar symptomatology as in pigs. Despite the economic loss in the pork industry due to S. suis infection and its importance as emerging zoonotic agent, experimental studies of S. suis virulence and pathology have been hampered by the lack of efficient methods for genetic transformation and the lack of a simple, cost-effective model to investigate S. suis virulence.
In some streptococcal species, genetic transformation can be carried out very efficiently as these species can be experimentally induced to take-up and recombine homologous extracellular DNA. The discovery of natural competence in some streptococci and the potential of opening up new avenues for genetic analysis of S. suis, was the motivation for investigating natural competence in this important pathogen.
In Chapter 2 we showed that a peptide pheromone induces competence in S. suis. The induction was dependent on ComX, a sigma factor that controls the streptococcal late competence regulon; the SigX-inducing peptide (XIP); and ComR, a regulator of comX. XIP was identified as an N-terminally truncated variant of ComS. This has resulted in the development of a novel methodology that will enable diverse research groups to accelerate discovery of novel features of S. suis ecology and pathology, especially with respect to virulence.
In Chapter 3 we investigated the genetic regulation of competence in S. suis and we provided a hypothetical model of the S. suis transformasome. We verified the essential role of the S. suis major pilin, and CinA for efficient competence development, supporting the notion that our predicted multi-protein transformasome indeed appears to function as described for other streptococci. We have also characterised the differential metabolic states that enable competence, and the metabolic state associated with competence exit (Chapter 4).
In Chapter 5 we investigated for the first time the use a zebrafish larvae model to assess the relative virulence of S. suis strains in porcine infections. Because of its convenience and cost-effectiveness, this model may be used to assay virulence of environmental S. suis strains, in particularly those of clinical relevance to infection of pigs and humans. Furthermore, a large number of bacterial mutants and strains can be screened for their virulence and in vivo pathogenicity, opening up new avenues to investigate the so far undiscovered pathways mediating successful host infection by S. suis.
In Chapter 6 we applied these two innovative methods (the competence system and the zebrafish larval model) to characterize two different two-component systems (TCS) of S. suis. TCS are important players in the regulation of bacterial adaptation to changes in environmental conditions, including those encountered in the host during infection. In this study, we studied the role of the two TCS of S. suis 2 strain S10 in virulence and in the survival of the bacteria in the bloodstream and host tissue.
Chapter 7 summarizes and discusses the key results and the future prospective of the thesis research.
Praktijk past onderzoeksresultaten Green challenge meeldauw gretig toe : onderzoek meeldauwbeheersing wegens succes verbreed
Staalduinen, J. van; Hofland-Zijlstra, J.D. - \ 2015
Onder Glas 12 (2015)4. - p. 36 - 37.
glastuinbouw - meeldauw - plantenziekten - ziektebestrijdende teeltmaatregelen - infectie - controle - systemische werking - vermeerderingsmateriaal - potplanten - resistentieveredeling - landbouwkundig onderzoek - greenhouse horticulture - mildews - plant diseases - cultural control - infection - control - systemic action - propagation materials - pot plants - resistance breeding - agricultural research
Onder de noemer Green Challenge Meeldauw startte vorig jaar een door het Productschap Tuinbouw en bedrijfsleven gefinancierd en door LTO Glaskracht Nederland begeleid masterplan, dat meer inzicht en oplossingen moet bieden bij het voorkomen en bestrijden van echte meeldauw. De aanvankelijke focus op siergewassen is inmiddels verbreed met vruchtgroenten. Onderzoekster Jantineke Hofland-Zijlstra van Wageningen UR Glastuinbouw doet verslag van de voorlopige resultaten.
Mating type and sexual fruiting body of Botrytis elliptica, the causal agent of fire blight in lily
Terhem, R.B. ; Staats, M. ; Kan, J.A.L. van - \ 2015
European Journal of Plant Pathology 142 (2015)3. - ISSN 0929-1873 - p. 615 - 624.
cinerea - leaves - resistance - infection - behavior - system
Botrytis elliptica is a necrotrophic pathogen that specifically infects Lilium species. Previous records show that B. elliptica collected in the field can successfully develop apothecia in vitro, however, there are no formal descriptions of apothecia of B. elliptica. The aim of this study was to analyse the sequence of the mating type loci of B. elliptica and produce apothecia in the laboratory in order to describe their morphology. The sequences of both MAT alleles (MAT1-1 or MAT1-2) of B. elliptica were determined and compared to the sister taxa, Botrytis cinerea and Sclerotinia sclerotiorum. Two strains of each mating type were used in crosses under controlled conditions to produce apothecia. Primordium rupture from sclerotial tissue occurred 74 days after fertilization and a mature apothecium formed within 1 month after rupture. The apothecia are 7 to 12 mm in height with a disk of 3 to 4 mm in diameter and 0.5 to 1 mm in thickness. The apothecial disk is usually umbilicate, depressed to funnel and rounded in shape. The number of apothecia growing on a sclerotium was one to nine. Asci are long, cylindrical with a size of 208¿×¿14 µm, thin walled and bearing eight ascospores. Ascospores are hyaline in colour, ellipsoidal with rounded ends, usually 18 to 24 µm in length and 6 to 10 µm in width (mean 19.5¿×¿8 µm). Ascospores were infectious on lily leaves.
Chikungunya virus non-structural protein 2-mediated host shut-off disables the unfolded protein response
Fros, J.J. ; Major, L.D. ; Scholte, F.E. ; Gardner, J. ; Hemert, M.J. van; Suhrbier, A. ; Pijlman, G.P. - \ 2015
Journal of General Virology 96 (2015)3. - ISSN 0022-1317 - p. 580 - 589.
endoplasmic-reticulum stress - semliki-forest-virus - messenger-rna - mammalian-cells - er stress - translational shutoff - transcription factor - gene-expression - insect cells - infection
The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1a become activated. PERK phosphorylates eIF2a leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1a splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2a and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.
Towards an Integrated Use of Biological Control by Cladosporium cladosporioides H39 in Apple Scab (Venturia inaequalis) Management
Köhl, J. ; Scheer, C. ; Holb, I.J. ; Masny, S. ; Molhoek, W.M.L. - \ 2015
Plant Disease 99 (2015)4. - ISSN 0191-2917 - p. 535 - 543.
potential biocontrol agents - leaf-litter - ascospore production - chaetomium-globosum - pathogen - resistance - antagonism - orchards - fungicides - infection
Apple scab, caused by Venturia inaequalis, is the most important disease in apple production, reducing yield and quality of fruit. Control of apple scab in commercial orchards currently depends on multiple applications of fungicides. The potential of the antagonistic isolate Cladosporium cladosporioides H39, originating from a sporulating colony of V. inaequalis, to control apple scab development was tested in eight trials during 2 years in orchards in Eperjeske (Hungary), Dabrowice (Poland), and Bavendorf (Germany) planted with different cultivars. Treatments were conducted as calendar sprays or after infection periods. Additional trials in an orchard in Randwijk (The Netherlands) focused on the effect of timing of antagonist application before or after infection periods. The overall results of the field trials consistently showed—for the first time—that stand-alone applications of the antagonist C. cladosporioides H39 can reduce apple scab in leaves and fruit. This was demonstrated in an organic growing system as well as in conventional orchards by spray schedules applied during the primary or the summer season. In both systems, the same control levels could be reached as with common fungicide schedules. Efficacies reached 42 to 98% on leaf scab incidence and 41 to 94% on fruit scab. The antagonist was also effective if applied one or even several days (equivalent to approximately 300 to 2,000 degree h) after infection events in several field trials and a trial conducted in Randwijk with single-spray applications at different intervals before or after infection events. Better understanding of the biology of the antagonist will help to further exploit its use in apple scab control
Cell-Free Propagation of Coxiella burnetii Does Not Affect Its Relative Virulence
Kuley, R. ; Smith, H.E. ; Frangoulidis, D. ; Smits, M.A. ; Roest, H.I.J. ; Bossers, A. - \ 2015
PLoS ONE 10 (2015)3. - ISSN 1932-6203 - 16 p.
real-time pcr - q-fever - ethidium monoazide - phase-ii - propidium monoazide - dead cells - t-cells - lipopolysaccharide - infection - strains
Q fever is caused by the obligate intracellular bacterium Coxiella burnetii. In vitro growth of the bacterium is usually limited to viable eukaryotic host cells imposing experimental constraints for molecular studies, such as the identification and characterisation of major virulence factors. Studies of pathogenicity may benefit from the recent development of an extracellular growth medium for C. burnetii. However, it is crucial to investigate the consistency of the virulence phenotype of strains propagated by the two fundamentally different culturing systems. In the present study, we assessed the viability of C. burnetii and the lipopolysaccaride (LPS) encoding region of the bacteria in both culture systems as indirect but key parameters to the infection potential of C. burnetii. Propidium monoazide (PMA) treatment-based real-time PCR was used for enumeration of viable C. burnetii which were validated by fluorescent infectious focus forming unit counting assays. Furthermore, RNA isolated from C. burnetiipropagated in both the culture systems was examined for LPS-related gene expression. All thus far known LPS-related genes were found to be expressed in early passages in both culturing systems indicating the presence of predominantly the phase I form of C. burnetii. Finally, we used immune-competent mice to provide direct evidence, that the relative virulence of different C. burnetii strains is essentially the same for both axenic and cell-based methods of propagation.
Baculovirus-induced tree-top disease: how extended is the role of egt as a gene for the extended phenotype?
Ros, V.I.D. ; Houte, S. van; Hemerik, L. ; Oers, M.M. van - \ 2015
Molecular Ecology 24 (2015)1. - ISSN 0962-1083 - p. 249 - 258.
spodoptera-exigua larvae - udp-glucosyl transferase - escherichia-coli - lepidopteran host - trichoplusia-ni - deletion - nucleopolyhedrovirus - behavior - insect - infection
Many parasites alter host behaviour to enhance their chance of transmission. Recently, the ecdysteroid UDP-glucosyl transferase (egt) gene from the baculovirus Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) was identified to induce tree-top disease in L. dispar larvae. Infected gypsy moth larvae died at elevated positions (hence the term tree-top disease), which is thought to promote dissemination of the virus to lower foliage. It is, however, unknown whether egt has a conserved role among baculoviruses in inducing tree-top disease. Here, we studied tree-top disease induced by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) in two different host insects, Trichoplusia ni and Spodoptera exigua, and we investigated the role of the viral egt gene therein. AcMNPV induced tree-top disease in both T. ni and S. exigua larvae, although in S. exigua a moulting-dependent effect was seen. Those S. exigua larvae undergoing a larval moult during the infection process died at elevated positions, while larvae that did not moult after infection died at low positions. For both T. ni and S. exigua, infection with a mutant AcMNPV lacking egt did not change the position where the larvae died. We conclude that egt has no highly conserved role in inducing tree-top disease in lepidopteran larvae. The conclusion that egt is a ‘gene for an extended phenotype’ is therefore not generally applicable for all baculovirus–host interactions. We hypothesize that in some baculovirus–host systems (including LdMNPV in L. dispar), an effect of egt on tree-top disease can be observed through indirect effects of egt on moulting-related climbing behaviour.
Transmission of white spot syndrome virus (WSSV) from Dendronereis spp. (Peters) (Nereididae) to penaeid shrimp
Haryadi, D. ; Verreth, J.A.J. ; Verdegem, M.C.J. ; Vlak, J.M. - \ 2015
Journal of Fish Diseases 38 (2015). - ISSN 0140-7775 - p. 419 - 428.
litopenaeus-vannamei - viral accommodation - baculovirus wsbv - scylla-serrata - mud crab - monodon - pathogenicity - host - infection - virulence
Dendronereis spp. (Peters) (Nereididae) is a common polychaete in shrimp ponds built on intertidal land and is natural food for shrimp in traditionally managed ponds in Indonesia. White spot syndrome virus (WSSV), an important viral pathogen of the shrimp, can replicate in this polychaete (Desrina et al. ); therefore, it is a potential propagative vector for virus transmission. The major aim of this study was to determine whether WSSV can be transmitted from naturally infected Dendronereis spp. to specific pathogen-free (SPF) Pacific white shrimp Litopenaeus vannamei (Boone) through feeding. WSSV was detected in naturally infected Dendronereis spp. and Penaeus monodon Fabricius from a traditional shrimp pond, and the positive animals were used in the current experiment. WSSV-infected Dendronereis spp. and P. monodon in a pond had a point prevalence of 90% and 80%, respectively, as measured by PCR. WSSV was detected in the head, gills, blood and mid-body of Dendronereis spp. WSSV from naturally infected Dendronereis spp was transmitted to SPF L. vannamei and subsequently from this shrimp to new naïve-SPF L. vannamei to cause transient infection. Our findings support the contention that Dendronereis spp, upon feeding, can be a source of WSSV infection of shrimp in ponds.
Schmallenberg Virus in Culicoides Biting Midges in the Netherlands in 2012
Elbers, A.R.W. ; Meiswinkel, R. ; Weezep, E. van; Kooi, E.A. ; Poel, W.H.M. van der - \ 2015
Transboundary and Emerging Diseases 62 (2015)3. - ISSN 1865-1674 - p. 339 - 342.
cattle - surveillance - bluetongue - infection - spp.
A total of 130 pools of Culicoides biting midges collected between May and September 2012 in the Netherlands were assayed for Schmallenberg virus (SBV). The Culicoides midges were caught in the same area as where in 2011 a high proportion of Culicoides pools tested positive for SBV, in majority with a high viral load (Ct values between 20 and 30). Two of a total of 42 pools comprising 50 midges/pool of the Obsoletus complex from the 2012 collection tested weak positive (Ct values: 34.96 and 37.66), indicating a relatively low viral load. On an individual midge level, the proportion of SBV-infected Culicoides of the Obsoletus complex caught in the same area and in a comparable period of the year was significantly lower in 2012 (0.1% = 1 per 1050 tested) compared with 2011 (0.56% = 13 per 2300 tested).
Latest developments on Streptococcus suis: an emerging zoonotic pathogen: part 1
Segura, M. ; Zheng, H. ; Greeff, A. de; Gao, G.F. ; Grenier, D. ; Jiang, Y. ; Chengping, L. ; Maskell, D. ; Oishi, K. ; Okura, M. ; Osawa, R. ; Schultsz, C. ; Schwerk, C. ; Sekizaki, T. ; Smith, H. ; Srimanote, P. ; Takamatsu, D. ; Tang, J. ; Tenenbaum, T. ; Tharavichitkul, P. ; Hoa, N.T. ; Valentin-Weigand, P. ; Wells, J.M. ; Wertheim, H. ; Zhu, B. ; Xu, J. ; Gottschalk, M. - \ 2014
Future Microbiology 9 (2014)4. - ISSN 1746-0913 - p. 441 - 444.
serotype-2 - thailand - infection - diversity
The first international workshop on Streptococcus suis, which is an important swine pathogen and emerging zoonotic agent, took place in Beijing, jointly organized by the Faculty of Veterinary Medicine, University of Montreal, Canada and the National Institute for Communicable Disease Control and Prevention, China CDC. The aim of the meeting was to gather together, for the first time, more than 80 researchers working on S. suis, from countries including China, Canada, Japan, The Netherlands, Germany, Thailand, the UK and Vietnam. This article, the first of a two-part report on this First International Workshop, reviews current aspects of the epidemiology and population genomics of S. suis, covers public health concerns and discusses questions about S. suis serotyping and molecular diagnostics.
Are the specialized bird ticks, Ixodes arboricola and I. frontalis, competent vectors for Borrelia burgdorferi sensu lato?
Heylen, D. ; Sprong, H. ; Oers, K. van; Fonville, M. ; Leirs, H. ; Matthysen, E. - \ 2014
Environmental Microbiology 16 (2014)4. - ISSN 1462-2912 - p. 1081 - 1089.
lyme-disease spirochete - tit parus-major - ricinus ticks - anaplasma-phagocytophilum - avian reservoir - passerine bird - acari ixodidae - central-europe - turdus-merula - infection
Our study tested whether two European bird-specialized ticks, Ixodes arboricola and I. frontalis, can act as vectors in the transmission cycles of Borrelia burgdorferi s.l. The ticks have contrasting ecologies but share songbird hosts (such as the great tit, Parus major) with the generalist I. ricinus which may therefore act as a bridging vector. In the first phase of the experiment, we obtained Borrelia-infected ornithophilic nymphs by exposing larvae to great tits that had previously been exposed to I. ricinus nymphs carrying a community of genospecies (Borrelia garinii, valaisiana, afzelii, burgdorferi s.s., spielmanii). Skin samples showed that birds selectively amplified B. garinii and B. valaisiana. The spirochetes were transmitted to the ornithophilic ticks and survived moulting, leading to infection rates of 16% and 27% in nymphs of I. arboricola and I. frontalis respectively. In the second phase, pathogen-free great tits were exposed to the Borrelia-infected ornithophilic nymphs. None of these ticks were able to infect the birds, as indicated by the tissue samples. Analysis of xenodiagnostic I. ricinus larvae found no evidence for co-feeding or systemic transmission of B. burgdorferi s.l. These outcomes do not support the occurrence of enzootic cycles of Borrelia burgdorferi s.l. involving songbirds and their specialized ornithophilic ticks.
Post-Travel Screening of Asymptomatic Long-Term Travelers to the Tropics for Intestinal Parasites Using Molecular Diagnostics
Soonawala, D. ; Lieshout, L. ; Boer, M.A.M. den; Claas, E.C.J. ; Verweij, J.J. ; Godkewitsch, A. ; Ratering, M. ; Visser, L.G. - \ 2014
American Journal of Tropical Medicine and Hygiene 90 (2014)5. - ISSN 0002-9637 - p. 835 - 839.
real-time pcr - strongyloides-stercoralis - laboratory tests - fecal samples - netherlands - microscopy - infection - diarrhea - protozoa
The incidence of asymptomatic travel-related parasitic infection is uncertain. Previous studies did not distinguish new incident infections, from past infections. Regardless of symptoms, we performed multiplex real-time polymerase chain reaction on pre- and post-travel stool samples of Dutch long-term travelers to the (sub)tropics. Serological screening for Schistosoma spp. was only performed in travelers to sub-Saharan Africa. In total, 679 travelers were included in the study. The follow-up rate was 82% (556 of 679). Participants' median travel duration was 12 weeks. There was one incident infection with Strongyloides stercoralis; there were none with Entamoeba histolytica, 4 with Cryptosporidium spp. (1%), and 22 with Giardia lamblia (4%). Nine of 146 travelers (6%) seroconverted for Schistosoma spp. Routine screening of stool samples for parasitic infection is not indicated for asymptomatic people, who travel to the (sub)tropics for up to 3 months. Screening for Schistosoma spp. should be offered to travelers with fresh-water contact in endemic regions.