Avirulent Strain Research Articles

The Cytokinin Complex Associated With Rhodococcus fascians: Which Compounds Are Critical for Virulence?

Virulent strains of Rhodococcus fascians cause a range of disease symptoms, many of which can be mimicked by application of cytokinin. Both virulent and avirulent strains produce a complex of cytokinins, most of which can be derived from tRNA degradation. To test the three current hypotheses regarding the involvement of cytokinins as virulence determinants, we used PCR to detect specific genes, previously associated with a linear virulence plasmid, including two methyl transferase genes (mt1 and mt2) and fas4 (dimethyl transferase), of multiple strains of R. fascians. We inoculated Pisum sativum (pea) seeds with virulent and avirulent strains of R. fascians, monitored the plants over time and compared these to mock-inoculated controls. We used RT-qPCR to monitor the expression of mt1, mt2, and fas4 in inoculated tissues and LC-MS/MS to obtain a comprehensive picture of the cytokinin complement of inoculated cotyledons, roots and shoots over time. The presence and expression of mt1 and mt2 was associated with those strains of R. fascians classed as virulent, and not those classed as avirulent. Expression of mt1, mt2, and fas4 peaked at 9 days post-inoculation (dpi) in cotyledons and at 15 dpi in shoots and roots developed from seeds inoculated with virulent strain 602. Pea plants inoculated with virulent and avirulent strains of R. fascians both contained cytokinins likely to have been derived from tRNA turnover including the 2-methylthio cytokinins and cis-zeatin-derivatives. Along with the isopentenyladenine-type cytokinins, the levels of these compounds did not correlate with virulence. Only the novel 1- and 2-methylated isopentenyladenine cytokinins were uniquely associated with infection by the virulent strains and are, therefore, the likely causative factors of the disease symptoms.

Cytokine expression in chicken embryo fibroblasts in response to infection with virulent or lentogenic avian avulavirus 1 (AAvV-1)

To investigate cytokine expression in chicken embryo fibroblast (CEF) cells, a virulent avian avulavirus 1 (AAvV-1) strain called SG10 that rapidly causes 100% mortality in its host, and a vaccine strain (La Sota) were characterized. Real-time quantitative PCR was performed on RNA samples from CEF cells, which were collected at 0, 24, 48 and 72 h post-infection. The dynamic expression patterns of ten cytokines (TNF-α, IFN-α, IFN-β, IL-1β, IL-2, IL-6, IL-10, IL-13, IL-15 and IL-18) were investigated. The results showed that infection with lentogenic La Sota induced significantly higher levels of the antiviral cytokines IFN-α and IFN-β, proinflammatory cytokines IL-2, IL-15 and IL-18, and the anti-inflammatory cytokine IL-10, than did infection with virulent SG10. Furthermore, the SG10 strain induced dramatically higher levels of the inflammatory cytokine IL-6 than those observed in cells infected with La Sota. However, the expression patterns of the other cytokines that were tested did not show any obvious trends or statistically significant differences between cells infected with the virulent and avirulent strains. These data show that infection with lentogenic La Sota induced more effective immune responses and anti-viral effects than did infection with virulent SG10 in CEFs. Our data provide distinct expression patterns of IFNs and proinflammatory and anti-inflammatory cytokines to AAvV-1 by virulence in CEF cells.

Deteksi Molekuler Gen Fusion (F) dan Analisis Perbandingan Beberapa Enzim Restriksi sebagai Penentu Patotipe Virus Newcastle Disease

Newcastle disease (ND) is a contagious viral disease caused by Avian Paramyxovirus Serotype-1 (APMV-1). This viral infection is responsible for devastating outbreak by attacking nerve, respiration, and also digestive system. This disease often followed with decreasing of eggs production and also responsible for economic losses in the poultry industries around the globe. The main goal was to differentiate virulent or avirulent strain of ND virus from F gene, which is the virulent marker of ND virus, by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Restrion Enzyme Analysis using BamH1, Hin 1l, and Apa 1. Ten ND virus samples came from Animal Disease Investigation Center (ADIC) Wates virus collection, collected from field case in 2012-2013. Newcastle disease virus was collected by extraction from the samples. The RNA product of extraction were used as a template for amplification in RT-PCR. The target of RT-PCR amplification was F gene. The results indicated positive reaction due to existing of DNA fragment band in size of 767 bp. RT-PCR and Restrion Enzyme Analysis can be used as tool to determine the pathotype of ND virus showed different restriction visualized by gel agarose electrophoresis.

Effectiveness of in planta control measures for Xylellafastidiosa.

This opinion updates the information included in the previous EFSA Scientific Opinion concerning the in planta control measures for Xylella fastidiosa, with a systematic review and critical analysis of the potential treatment solutions that have been published against this pest so far. The output of this opinion focuses on the application of chemical or biological treatments on living plants. In vitro studies, hot water treatments, use of resistant varieties and vector control are excluded from the review. The use of antibiotics is not considered due to the risk of antimicrobial resistance development. The use of weakly virulent or avirulent strains of X. fastidiosa is covered in this review, although this organism is an EU quarantine plant pest and its introduction in the EU territory is banned. Experiments were recently conducted to assess the effect of application of zinc, copper, and citric acid biocomplex, of N‐acetylcysteine, and of ‘diffusible signal factor’ (and of its homologs). Their results showed that these control measures were sometimes able to reduce symptoms caused by X. fastidiosa. Recent experiments also showed that several species of endophytic microorganisms, some bacteriophages and inoculation of weakly virulent/avirulent strains of X. fastidiosa could offer some protection against the Pierce's disease. However, based on the reviewed results, the Panel concludes that, although several published experiments show some effects in reducing symptoms development, the tested control measures are not able to completely eliminate X. fastidiosa from diseased plants. The Panel confirms as previously stated that there is currently no control measure available to eliminate the bacteria from a diseased plant in open field conditions.

Effect of Oxygen Concentration on Susceptibility of Neisseria Species to Resazomycins

A growing number of human pathogens are becoming resistant to most common antibiotics. Each year, antibiotic-resistant infections are responsible for 23,000 deaths in the United States and billions of dollars in health care costs. To prevent the loss of additional lives from once “curable” diseases, new antibiotics must be developed. We recently identified a novel family of resazurin-based compounds, resazomycins, which exhibit antimicrobial activity against various Neisseria species including the human pathogen N. gonorrhoeae in vitro. However, when these compounds were tested in a mouse model of gonorrhea, few resazomycins exhibited any therapeutic effect. These differences in in vitro and in vivo therapeutic efficacy may be due to differences in oxygen concentration. Most mammalian tissues exist at oxygen concentrations well below atmospheric levels, typically 3-5% instead of 20%. Therefore, we hypothesized that decreased oxygen levels may confer resistance to resazomycins. To test this hypothesis, we cultivated the avirulent Neisseria strain, N.sicca, under atmospheric (20%) and physiologic (2-3%) oxygen conditions and then measured the susceptibility of the bacteria to various resazomycins. To date, more N. sicca growth was observed following exposure to resazurin and resorufin under physiologic oxygen than atmospheric oxygen suggesting oxygen levels effect susceptibility to resazomycins. Further investigation is needed to determine the precise mechanism by which N. sicca is becoming resistant to resazomycins under low oxygen conditions.

Genetic buffering of cyclic AMP in Arabidopsis thaliana compromises the plant immune response triggered by an avirulent strain of Pseudomonas syringae pv. tomato.

Cyclic AMP plays important roles in different physiological processes, including plant defence responses. However, as little information is known on plant enzymes responsible for cAMP production/degradation, studies of cAMP functions have relied, to date, on non-specific pharmacological approaches. We therefore developed a more reliable approach, producing transgenic Arabidopsis thaliana lines overexpressing the 'cAMP-sponge' (cAS), a genetic tool that specifically buffers cAMP levels. In response to an avirulent strain of Pseudomonas syringae pv. tomato (PstAvrB), cAS plants showed a higher bacterial growth and a reduced hypersensitive cell death in comparison with wild-type (WT) plants. The low cAMP availability after pathogen infection delayed cytosolic calcium elevation, as well as hydrogen peroxide increase and induction of redox systems. The proteomic analysis, performed 24h post-infection, indicated that a core of 49 proteins was modulated in both genotypes, while 16 and 42 proteins were uniquely modulated in WT and cAS lines, respectively. The involvement of these proteins in the impairment of defence response in cAS plants is discussed in this paper. Moreover, in silico analysis revealed that the promoter regions of the genes coding for proteins uniquely accumulating in WT plants shared the CGCG motif, a target of the calcium-calmodulin-binding transcription factor AtSR1 (Arabidopsis thaliana signal responsive1). Therefore, following pathogen perception, the low free cAMP content, altering timing and levels of defence signals, and likely acting in part through the mis-regulation of AtSR1 activity, affected the speed and strength of the immune response.

Characterisation of genes differentially expressed in macrophages by virulent and attenuated Mycobacterium tuberculosisthrough RNA-Seq analysis

Tuberculosis (TB) remains a global healthcare issue. Understanding the host-pathogen interactions in TB is vital to develop strategies and therapeutic tools for the control of Mycobacterium tuberculosis (Mtb). In this study, transcriptome analyses of macrophages infected with either the virulent Mtb strain H37Rv (Rv) or the avirulent Mtb strain H37Ra (Ra) were carried out and 750 differentially expressed genes (DEGs) were identified. As expected, the DEGs were mainly involved in the induction of innate immune responses against mycobacterial infections. Among the DEGs, solute carrier family 7 member 2 (Slc7a2) was more strongly expressed in Ra-infected macrophages. Induction of SLC7A2 was important for macrophages to control the intracellular survival of Mtb. Our results imply that SLC7A2 plays an important role in macrophages during Mtb infection. Our findings could prove useful for the development of new therapeutic strategies to control TB infection.

Adherent/invasive capacities of bovine-associated Aerococcus viridans contribute to pathogenesis of acute mastitis in a murine model

Aerococcus viridans, a firmicutes bacteria widespread in the environment, is increasingly isolated from humans and animals, especially cows with mastitis. However, its pathogenicity in the bovine mammary gland is unclear. The objective was to explore pathogenic potential of putative virulent and avirulent A. viridans in murine systemic and intramammary infection and mechanistically in cultured bovine mammary epithelial cells (bMECs). Virulence of 9 strains of A. viridans, isolated from subclinical cases of mastitis, was tested for their ability to kill mice when systemically inoculated. Two A. viridans strains, causing highest and lowest survival rate in mice, were selected further as putative avirulent and virulent strains, respectively. Staphylococcus aureus N305 was used as a positive control. After intramammary inoculation, the virulent strain survived and replicated in the murine mammary gland for 9 d, whereas the avirulent strain was eliminated within 3 d. The virulent strain induced a robust inflammatory reaction in the mammary gland, characterized by acute histopathological changes, increased myeloperoxidase activity and higher expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) compared to the avirulent strain. The virulent strain produced CAMP factor and exhibited strong cytotoxic effects (LDH release) and adhering and invasive abilities in contact with bMECs. Adhesion and invasion of virulent strain to bMECs was further confirmed by scanning and transmission electron microscopy; there was severe damage, including cytomembrane disruption, swollen mitochondria and loss of organelles. In conclusion, the putative virulent strain of A. viridans activated a strong neutrophil-based inflammatory response in the mammary gland, attributed to its ability to adhere to and invade mammary epithelium.

Abstract A170: Transient microbiota depletion enhances mucosal CD8 T-cell responses

Abstract CD8+ tissue resident memory T-cells (Trm) are poised for immediate reactivation in peripheral organs and have enhanced protective potential in comparison to circulating T-cells. Trm represent potential players in antitumor immunity that might be enhance the effectiveness of cancer vaccines. Vaccination strategies increasing accumulation of Trm cells in skin or in the genital tract augmented protection against tissue-specific pathogens in vivo. However, to date, no specific approach has been proposed to reinforce Trm in the intestinal mucosa. The microbiota shapes composition and functions of host immune populations, particularly in the intestinal tissue, where it determines the ratio between conventional and regulatory T-cells. This suggests that modulation of commensal communities might be exploited to augment numbers and function of gut-resident CD8 T-cells. Listeria monocytogenes attenuated strains are employed in clinical trials to promote immunity against tumor antigens and have been administered orally to healthy volunteers to promote mucosal immunity, proving safety of this approach. Developing more effective mucosal vaccination strategies based on oral administration of Listeria-based vectors, would have a great impact in the fight against tumors of the gastrointestinal tract, such as colorectal cancer, for which neoantigens are being discovered. Based on our recent observation that antibiotic treatment favors intestinal expansion of Listeria, we have devised an oral immunization strategy (TMDI, Transient Microbiota Depletion prior to Immunization) consisting of oral vaccination with an antigen-carrying Listeria strain following administration of a single dose of antibiotics. In mice, TMDI promoted expansion of engineered avirulent Listeria strains, resulting in striking expansion of the antigen-specific CD8+ Trm population (~20 fold), that additionally displayed potent effector functions. Mice immunized with this approach showed augmented protection upon rechallenge with high doses of pathogen.We found that increased accumulation of T-cells was dependent on both increased antigen load and stronger inflammatory stimuli; surprisingly, even when the vector was forcedly maintained at high levels in the intestinal lumen for several weeks, the elicited CD8+ Trm cells did not show any sign of exhaustion and retained the capacity to rapidly produce inflammatory cytokines upon antigen stimulation. In conclusion, we have identified a novel strategy to harness tissue resident CD8 T-cells against an epitope of choice by modulating microbiota density at the time of immunization, and set up a system to dissect the requirements for obtaining potent T-cell responses in the intestine. This tool can potentially be utilized to design improved vaccines against tumors of the intestinal tract. Citation Format: Simone Becattini, Alexander Louie, Ingrd M. Leiner, Eric Pamer. Transient microbiota depletion enhances mucosal CD8 T-cell responses [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A170.

In vitro growth kinetics and gene expression analysis of the turkey adenovirus 3, a siadenovirus

Turkey adenovirus 3 (TAdV-3) belongs to the genus Siadenovirus, family Adenoviridae. Previously, nucleotide sequencing and annotation of the Virginia avirulent strain (VAS) of TAdV-3 genome, isolated in our laboratory, indicated the presence of a total of 23 genes and open reading frames (ORFs). The goals of this study were 1) to delineate the growth kinetics of the virus using a qPCR-based infectivity assay, and 2) to determine the virus gene expression profile during the early and late phases of infection in target B lymphocytes. The one-step growth curve experiment demonstrated 3 phases of virus replication cycle: a lag phase lasted for 12–18 h post-infection (h.p.i.), in which the virus titer declined; a log phase from 18 to 120 h.p.i., in which the number of infectious virus particles increased over 20,000 folds, and a brief decline phase thereafter. Southern blot analysis indicated that the synthesis of new viral DNA started by 8 h.p.i. Gene-specific RT-PCR analysis revealed the expression of mRNAs from the 23 TAdV-3 genes/ORFs. According to the temporal transcriptional profiling of TAdV-3 genome, genes could be divided into 3 groups based on the time of transcription initiation: group 1 showed detectable levels of transcription at 2 h.p.i and included 7 genes, i.e., hyd, III, pX, pVI, II, 100 K, and 33 K; group 2 included 12 genes whose mRNAs were detected for the first time at 4 h.p.i., i.e., ORF1, IVa2, pol, pTP, pIIIa, EP, DBP, E3, U exon, IV, ORF7, and ORF8; group 3 of transcripts were detectable starting 8 h.p.i. and included only 4 genes, i.e., 52 K, 22 K, pVII, and pVIII. Our data suggest that the transcriptional kinetics of genus Siadenovirus differ from that observed in other adenoviral genera; however, a few TAdV-3 genes showed similar expression patterns to their adenoviral homologs.

Redox Proteome Perturbation in Arabidopsis upon Pseudomonas syringae Infection

Oxidative burst is one of the earliest plant cellular responses triggered by pathogen infection. Reactive oxygen species can cause oxidative modifications of redox-sensitive proteins to mediate the defense responses. Identification and characterization of proteins that undergo oxidative modifications in these processes is an important step toward understanding molecular mechanisms of plant defense responses. In this study, an in vivo 15N metabolic labeling method combined with a cysteine-containing peptide enrichment technique was applied to identify and quantify proteins and their redox states in Arabidopsis in response to infection by Pseudomonas syringae pv tomato DC3000 (Pst). Changes of peptide redox states were compared and corrected with the changes of protein levels. A total of forty peptides representing thirty-six non-redundant proteins showed significantly redox state changes in response to the infection by the virulent Pst strain and the avirulent Pst strain (Pst avrRpm1), of which 23 had previously not been recognized to undergo oxidative PTMs. The differentially expressed redox-sensitive proteins are involved in cell wall organization, primary metabolism, photosynthesis and stress responses. Interestingly, proteins located at extracellular were more susceptible to be regulated on the redox PTMs level. These findings provide a foundation for further investigation into the redox signaling during plant defense responses.

Active and inactive forms of biotin synthase occur in Heterodera glycines.

Heterodera glycines, the soybean cyst nematode (SCN), is a plant-parasitic nematode capable of manipulating host plant biochemistry and development. Many studies have suggested that the nematode has acquired genes from bacteria via horizontal gene transfer events (HGTs) that have the potential to enhance nematode parasitism. A recent allelic imbalance analysis identified two candidate virulence genes, which also appear to have entered the SCN genome through HGTs. One of the candidate genes, H. glycines biotin synthase (HgBioB), contained sequence polymorphisms between avirulent and virulent inbred SCN strains. To test the function of these HgBioB alleles, a complementation experiment using biotin synthase-deficient Escherichia coli was conducted. Here, we report that avirulent nematodes produce an active biotin synthase while virulent ones contain an inactive form of the enzyme. Moreover, sequencing analysis of HgBioB genes from SCN field populations indicates the presence of diverse mixture of HgBioB alleles with the virulent form being the most prevalent. We hypothesize that the mutations in the inactive HgBioB allele within the virulent SCN could result in a change in protein function that in some unknown way bolster its parasitic lifestyle.

RiceMetaSysB: a database of blast and bacterial blight responsive genes in rice and its utilization in identifying key blast-resistant WRKY genes.

Nearly two decades of revolution in the area of genomics serves as the basis of present-day molecular breeding in major food crops such as rice. Here we report an open source database on two major biotic stresses of rice, named RiceMetaSysB, which provides detailed information about rice blast and bacterial blight (BB) responsive genes (RGs). Meta-analysis of microarray data from different blast- and BB-related experiments across 241 and 186 samples identified 15135 unique genes for blast and 7475 for BB. A total of 9365 and 5375 simple sequence repeats (SSRs) in blast and BB RGs were identified for marker development. Retrieval of candidate genes using different search options like genotypes, tissue, developmental stage of the host, strain, hours/days post-inoculation, physical position and SSR marker information is facilitated in the database. Search options like ‘common genes among varieties’ and ‘strains’ have been enabled to identify robust candidate genes. A 2D representation of the data can be used to compare expression profiles across genes, genotypes and strains. To demonstrate the utility of this database, we queried for blast-responsive WRKY genes (fold change ≥5) using their gene IDs. The structural variations in the 12 WRKY genes so identified and their promoter regions were explored in two rice genotypes contrasting for their reaction to blast infection. Expression analysis of these genes in panicle tissue infected with a virulent and an avirulent strain of Magnaporthe oryzae could identify WRKY7, WRKY58, WRKY62, WRKY64 and WRKY76 as potential candidate genes for resistance to panicle blast, as they showed higher expression only in the resistant genotype against the virulent strain. Thus, we demonstrated that RiceMetaSysB can play an important role in providing robust candidate genes for rice blast and BB.

The N and P genes facilitate pathogenicity of the rabies virus G gene

To explore the effects of different gene combinations on the pathogenicity of the rabies virus (RABV), six chimeric RABV mutants, rRC-HL(G), rRC-HL(NG), rRC-HL(PG), rRC-HL(NP), rRC-HL(NM) and rRC-HL(NPG), were constructed using a reverse genetic technique based on an avirulent parental rRC-HL strain and a virulent parental GX074 isolate. These mutants were intracerebrally inoculated into adult mice. The results indicated that 10<sup>2</sup> ffu and 10<sup>6</sup> ffu of rRC-HL(G), rRC-HL(NG), rRC-HL(PG) and rRC-HL(NPG) were 100% lethal. In the case of intramuscular viral infection, none of the mice inoculated with 10<sup>2 </sup>ffu of any of the RABV mutants, including GX074, died; at 10<sup>6 </sup>ffu, rRC-HL(G) was lethal in 2/5 cases, rRC-HL(NG) was lethal in 1/5 cases and rRC-HL(PG) was lethal for 2/5 mice. The rRC-HL(NPG) mutant was fatal for 3/5 mice, as was the parental GX074. Furthermore, the LD<sub>50</sub> values of the chimeric RABV mutants were measured, with the results showing that the LD<sub>50</sub> values of both rRC-HL(NG) and rRC-HL(PG) were lower than that of rRC-HL(G), but higher than that of rRC-HL(NPG). Thus, the action of N + G, or P + G, or N + P + G gene combinations may be more pronounced than that of the G gene alone. Body weight changes and the clinical symptoms of the tested mice were consistent with pathogenicity. These data indicate that the N and P genes are involved in and facilitate the pathogenicity of the RABV G gene. These experiments provide further evidence that multi-gene cooperation is responsible for the virulence of RABV.

Co-expression of the Hemagglutinin and Neuraminidase by Heterologous Newcastle Disease Virus Vectors Protected Chickens against H5 Clade 2.3.4.4 HPAI Viruses

Avian influenza remains an important zoonotic disease with a significant global impact. The spread of H5 highly pathogenic avian influenza (HPAI) viruses (clade 2.3.4.4) by migratory birds has caused outbreaks in wide geographic regions (Asia, Europe, and North America) with great economic losses during 2014–2015. Efficient vaccines and vaccination approaches are needed to enhance protective immunity against HPAI viruses. Although several vaccination strategies have been developed, none has been satisfactory. Our strategy has been to use avirulent vaccine strain of Newcastle disease virus (NDV) as a vaccine vector for HPAI viruses. For poultry vaccination, we previously generated a new platform of chimeric NDV vector to overcome preexisting maternal antibodies to NDV in poultry. In this study, we have generated vaccine candidates targeting H5 clade 2.3.4.4 HPAI viruses by using our chimeric NDV and conventional NDV strain LaSota vectors for a heterologous prime-boost immunization approach. Co-expression of the HA and NA proteins by our vaccine vectors induced enhanced HPAI virus specific immune responses in specific-pathogen free and broiler chickens prior to challenge. Further, these vaccine candidates efficiently protected broiler chickens from mortality, clinical signs, and shedding of homologous and heterologous H5 HPAI viruses and highly virulent NDV, thus providing a dual vaccination approach in the field.

Curcumin nanoemulsion as a novel chemical for the treatment of acute and chronic toxoplasmosis in mice.

BackgroundThe aim of this study was to prepare curcumin nanoemulsion (CR-NE) to solve the problems associated with poor water solubility and low bioavailability of CR and to test its efficiency in the treatment of acute and chronic toxoplasmosis in mouse models.Materials and methodsCR-NE 1% was prepared using spontaneous emulsification by soybean as oil phase; a mixture of Tween 80 and Tween 85 as surfactant; ethanol as cosurfactant and distilled water. Particle size and zeta potential of NE were assessed using Nano-ZS90 dynamic light scattering. Stability testing of NE was assessed after storage for 2 months at room temperature. In vivo experiments were carried out using 50 BALB/c mice inoculated with virulent RH strain (type I) and 50 BALB/c mice inoculated with avirulent Tehran strain (type II) of Toxoplasma gondii and treated with CR-NE (1% w/v), CR suspension (CR-S, 1% w/v), and NE without CR (NE-no CR).ResultsThe mean particle size and zeta potential of CR-NE included 215.66±16.8 nm and −29.46±2.65 mV, respectively, and were stable in particle size after a three freeze–thaw cycle. In acute phase experiment, the survival time of mice infected with RH strain of T. gondii and treated with CR-NE extended from 8 to 10 days postinoculation. The differences were statistically significant between the survival time of mice in CR-NE-treated group compared with negative control group (P<0.001). Furthermore, CR-NE significantly decreased the mean counts of peritoneum tachyzoites from 5,962.5±666 in negative control group to 627.5±73 in CR-NE-treated mice (P<0.001). Growth inhibition rates of tachyzoites in peritoneum of mice receiving CR-NE, CR-S, and NE-no CR included 90%, 21%, and 11%, respectively, compared with negative control group. In chronic phase experiment, the average number and size of tissue cysts significantly decreased to 17.2±15.6 and 31.5±6.26 µm, respectively, in mice inoculated with bradyzoites of T. gondii Tehran strain and treated with CR-NE compared with that in negative control group (P<0.001). Decrease of cyst numbers was verified by downregulation of BAG1 in treatment groups compared with negative control group with a minimum relative expression in CR-NE (1.12±0.28), CR-S (11.76±0.87), and NE-no CR (14.67±0.77), respectively, (P<0.001).ConclusionResults from the current study showed the potential of CR-S and CR-NE in treatment of acute and chronic toxoplasmosis in mouse models for the first time. However, CR-NE was more efficient than CR-S, and it seems that CR-NE has a potential formula for the treatment of acute and chronic toxoplasmosis, especially in those with latent bradyzoites in brain.

CanTF, a Novel Transcription Factor in Pepper, Is Involved in Resistance to Phytophthora capsici as well as Abiotic Stresses

Stress is usually considered an important factor resulting in plant injury. In this study, we identified a novel transcription factor gene, CanTF (Capsicum annuum transcription factor IIB), and characterized its role in the response to biotic and abiotic stresses. The full-length CanTF cDNA consists of 1488 bp, with a 1281-nucleotide open reading frame (ORF), and encodes a protein containing 426 amino acids with a theoretical molecular weight of 46.84 kDa. Real-time quantitative PCR revealed that CanTF is a stress-induced gene, with increased expression levels under both biotic and abiotic stresses. The expression of CanTF in pepper organs, especially roots, was highly induced by inoculation with an avirulent strain of Phytophthora capsici. Additionally, the differential expression of CanTF was observed under abiotic stresses, i.e., earlier expression was detected after cold, drought, and SA treatments than after salt and H2O2 treatment, suggesting its role in responses to various abiotic stresses. Furthermore, the silencing of CanTF by virus-induced gene silencing reduced the expression of defense-related genes (CaPR1, CaDEF1, and CaSAR82) under P. capsici inoculation. POD and root activity levels were lower after gene silencing than in controls, demonstrating the positive regulatory effect of CanTF against P. capsici. These results suggested that CanTF is a stress-induced gene involved in strengthening the pepper defense against biotic and abiotic stresses.

Elements Involved in the Rsv3-Mediated Extreme Resistance against an Avirulent Strain of Soybean Mosaic Virus.

Extreme resistance (ER) is a type of R-gene-mediated resistance that rapidly induces a symptomless resistance phenotype, which is different from the phenotypical R-resistance manifested by the programmed cell death, accumulation of reactive oxygen species, and hypersensitive response. The Rsv3 gene in soybean cultivar L29 is responsible for ER against the avirulent strain G5H of soybean mosaic virus (SMV), but is ineffective against the virulent strain G7H. Rsv3-mediated ER is achieved through the rapid accumulation of callose, which arrests SMV-G5H at the point of infection. Callose accumulation, however, may not be the lone mechanism of this ER. Analyses of RNA-seq data obtained from infected soybean plants revealed a rapid induction of the abscisic acid pathway at 8 h post infection (hpi) in response to G5H but not to G7H, which resulted in the down-regulation of transcripts encoding β-1,3 glucanases that degrade callose in G5H-infected but not G7H-infected plants. In addition, parts of the autophagy and the small interfering (si) RNA pathways were temporally up-regulated at 24 hpi in response to G5H but not in response to G7H. The jasmonic acid (JA) pathway and many WRKY factors were clearly up-regulated only in G7H-infected plants. These results suggest that ER against SMV-G5H is achieved through the quick and temporary induction of ABA, autophagy, and the siRNA pathways, which rapidly eliminate G5H. The results also suggest that suppression of the JA pathway in the case of G5H is important for the Rsv3-mediated ER.

Recombinant infectious bronchitis viruses expressing heterologous S1 subunits: potential for a new generation of vaccines that replicate in Vero cells.

The spike glycoprotein (S) of infectious bronchitis virus (IBV) comprises two subunits, S1 and S2. We have previously demonstrated that the S2 subunit of the avirulent Beau-R strain is responsible for its extended cellular tropism for Vero cells. Two recombinant infectious bronchitis viruses (rIBVs) have been generated; the immunogenic S1 subunit is derived from the IBV vaccine strain, H120, or the virulent field strain, QX, within the genetic background of Beau-R. The rIBVs BeauR-H120(S1) and BeauR-QX(S1) are capable of replicating in primary chicken kidney cell cultures and in Vero cells. These results demonstrate that rIBVs are able to express S1 subunits from genetically diverse strains of IBV, which will enable the rational design of a future generation of IBV vaccines that may be grown in Vero cells.

Insights into the genome sequence of ovine Pasteurella multocida type A strain associated with pneumonic pasteurellosis

Pasteurella multocida is a causative agent of pneumonic pasteurellosis in ruminants. Bovine and porcine strains of P. multocida have been sequenced and analyzed worldwide. However limited genomic information is available on ovine strains involved in causation of pneumonic pasteurellosis. Therefore, the present study focused on genome sequencing and analysis of a P. multocida type A isolate recovered from respiratory infection of sheep to unravel the genetic contents and its involvement in the disease.P. multocida type A strain NIVEDI/PMS-1 genome was 2,543,931bp in size, assembled into 67 contigs, consisted of 1879 protein-coding regions, 19 genomic islands (GIs) and 10 phage regions. The identified GIs and phage regions showed high sequence identity mostly with other Pasteurellaceae family respiratory pathogens indicating horizontal gene transfer in ovine respiratory niche. The phylogenetic analysis revealed strain NIVEDI/PMS-1 as closest neighbor to P. multocida P1933 strain reported from bovine. Comparative genomic analysis of strain NIVEDI/PMS-1 with virulent GX-Pm and avirulent Pm70 strains revealed presence of 45 virulence genes common with virulent strain. Further, various virulence factors and accessory genes needed for survival and pathogenesis were also identified. Dermonecrotic toxin (ToxA) like protein presence in the strain appears interesting and further study is needed to understand its role in ovine pasteurellosis.