Different Levels Of Virulence Research Articles

Toxicity of some nanomaterials against Botrytis allii, the causal pathogen of neck rot disease of onion (Allium cepa L.)

In this study, 14 fungal isolates associated with symptomatic onion samples collected from different counties of Sohag Governorate showing neck rot disease were obtained and identified as Botrytis allii Munn (12 isolates) and B. cinerea Pers. (2 isolates). Pathogenicity tests carried out in the greenhouse showed that only isolates of B. allii were pathogenic to onion plants and caused identical neck rot symptoms among all fungal isolates tested. However, B. allii isolates exhibited different levels of virulence. Isolate No. 9 of B. allii was the high pathogenic one, where it caused 95 and 81.65% of disease incidence and severity, respectively. In vitro, the influence of carbon, MgO, and ZnO nanoparticles tested at 0.1, 0.2, and 0.3 mM on mycelial linear growth and biomass of B. allii was studied. Results obtained showed that all tested nanomaterials significantly reduced the mycelial linear growth and biomass at all concentrations tested. The nano MgO at 0.3 mM caused the high inhibitory effect followed by nano ZnO,

First Report on the Fungal Pathogens Associated with Leaf Tip Blight of Oil Palm at Nursery Stage in Malaysia

Oil palm has been contributing to the economic growth and gross national income (GNI) of Malaysia. On that note, supply of disease-free oil palm seedlings to field is crucial. At present many nurseries encounter disease infestations of the seedlings at nursery stages. One of the most common diseases identified was leaf tip blight. The disease incidences cause critical quality, quantity loss of the seedlings and draw other pathogens in the field to infect the initially infected host. Till to date, no study has been conducted to diagnose leaf tip blight disease of oil palm at nursery stage in Malaysia. Molecular identification was conducted by using 3 specific primers namely actin gene (ACT), betatubulin-2 (Bt2), glyceraldehyde-3-phosphate dehydrogenase (GD) gene and complete rDNA-ITS (ITS) to confirm the isolates. All the isolated fungi matched 98% to 100% similarity with GenBank database. Thus, the isolated isolates were identified as Nigrospora sp., Colletotrichum gloeosporioides and Phoma sp. In vitro pathogenicity test demonstrated that, the three-isolates produced similar leaf tip blight symptoms as the infested seedlings at the nursery. As a conclusion, Nigrospora sp., C. gloeosporioides and Phoma sp. were identified as the causal pathogen of leaf tip blight of oil palm with different levels of virulence.

Isolation and Characterization of Rhodococcus spp. from Pistachio and Almond Rootstocks and Trees in Tunisia

The purpose of this study was to isolate and identify Rhodococcus spp. strains from almond and pistachio rootstocks and trees in Tunisia. Twenty-eight strains were identified through 16S rDNA and vicA genes amplification and sequencing. Pea bioassay was performed to determine the pathogenicity of the strains. Representative 16S rDNA and vicA sequences of eight strains from pistachio and seven strains from almond were closely related (>98% similarity) to Rhodococcus spp. accessions in GenBank. Phylogenetic analysis based on 16S rDNA sequences revealed that the yellow-colored strains clustered with phytopathogenic Rhodococcusfascians. The red and orange-colored strains were separated into a different group with R. kroppenstedtii and R. corynebacteiroides isolates. Eleven strains affected the pea seedlings’ growth and exhibited different levels of virulence. The number of shoots was significantly higher in seedlings inoculated with four Rhodococcus strains, whereas the other three strains caused up to 80% of plant height reduction and reduced root secondary growth compared to non-inoculated pea seedlings. These strains, most of which are epiphytes from asymptomatic hosts, showed strong pathogenicity during pea bioassay and were established endophytically in pea tissues. Ten att and five fas genes were detected in four strains and may represent a novel model of plant pathogenic Rhodococcus virulence. The results of our survey showed that Rhodococcus is present but not prevalent in all visited orchards of almond and pistachio rootstocks and trees. Our surveys complemented the investments being made on ornamental species in Tunisia and unveiled the presence of undocumented plant-associated Rhodococcus spp. on economically important crops.

Whole-genome comparative analysis of Malaysian Burkholderia pseudomallei clinical isolates.

Burkholderia pseudomallei , a soil-dwelling Gram-negative bacterium, is the causative agent of the endemic tropical disease melioidosis. Clinical manifestations of B. pseudomallei infection range from acute or chronic localized infection in a single organ to fulminant septicaemia in multiple organs. The diverse clinical manifestations are attributed to various factors, including the genome plasticity across B. pseudomallei strains. We previously characterized B. pseudomallei strains isolated in Malaysia and noted different levels of virulence in model hosts. We hypothesized that the difference in virulence might be a result of variance at the genome level. In this study, we sequenced and assembled four Malaysian clinical B. pseudomallei isolates, UKMR15, UKMPMC2000, UKMD286 and UKMH10. Phylogenomic analysis showed that Malaysian subclades emerged from the Asian subclade, suggesting that the Malaysian strains originated from the Asian region. Interestingly, the low-virulence strain, UKMH10, was the most distantly related compared to the other Malaysian isolates. Genomic island (GI) prediction analysis identified a new island of 23 kb, GI9c, which is present in B. pseudomallei and Burkholderia mallei , but not Burkholderia thailandensis . Genes encoding known B. pseudomallei virulence factors were present across all four genomes, but comparative analysis of the total gene content across the Malaysian strains identified 104 genes that are absent in UKMH10. We propose that these genes may encode novel virulence factors, which may explain the reduced virulence of this strain. Further investigation on the identity and role of these 104 proteins may aid in understanding B. pseudomallei pathogenicity to guide the design of new therapeutics for treating melioidosis.

Molecular variation among virulent and avirulent strains of the quarantine nematode Bursaphelenchus xylophilus

Bursaphelenchus xylophilus is an emerging pathogenic nematode that is responsible for a devastating epidemic of pine wilt disease worldwide, causing severe ecological damage and economic losses to forestry. Two forms of this nematode have been reported, i.e., with strong and weak virulence, commonly referred as virulent and avirulent strains. However, the pathogenicity-related genes of B. xylophilus are not sufficiently characterized. In this study, to find pathogenesis related genes we re-sequenced and compared genomes of two virulent and two avirulent populations. We identified genes affected by genomic variation, and functional annotation of those genes indicated that some of them might play potential roles in pathogenesis. The performed analysis showed that both avirulent populations differed from the virulent ones by 1576 genes with high impact variants. Demonstration of genetic differences between virulent and avirulent strains will provide effective methods to distinguish these two nematode virulence forms at the molecular level. The reported results provide basic information that can facilitate development of a better diagnosis for B. xylophilus isolates/strains which present different levels of virulence and better understanding of the molecular mechanism involved in the development of the PWD.

Comparative analysis of two Orobanche cumana Wallr. accessions with a different virulence

Orobanche cumana Wallr. is a root parasite significantly affecting sunflower production in most cultivating countries. Due to the continuous introduction of resistant sunflower hybrids, the parasite evolves rapidly, developing new, more virulent races. In this context, extensive knowledge of the distinctive peculiarities of races and their pathogenicity is needed to understand the broomrape evolution and develop effective control strategies. The present study examines the comparative morphologic, genetic, transcriptomic, and proteomic analysis of two O. cumana accessions with different level of virulence (belonging to race E and H, respectively). Using simple sequence repeats (SSR) and inter-simple sequence repeats (ISSR) markers, specific amplicons suitable for distinguishing between races have been revealed. It has also been found that the genes PME, PGU, PRX, and CHS that encode enzymes associated with host invasion, such as pectin methylesterases, polygalacturonases, peroxidases, and chalcone synthases, respectively, were upregulated in race H compared to E at different developmental stages, confirming their role in the pathogenicity of broomrape. The proteomic analysis has showed 19 differentially expressed proteins (DEP), including 6 specific for race E and 13 specific for race H. A number of DEP were identified as enzymes of the respiratory system, cell wall-modifying enzymes, stress-related proteins, and other proteins known to be involved in the invasion of the host, and might be important for the more virulent race H. Obtained results provide new insight into broomrape parasitism and offer new opportunities for controlling this pathogen.

Tregsin the immune response of BALB/c mice experimentally infected with species of the Sporothrix genus.

Background: Sporotrichosis occurs through contact with contaminated soil and plant. However, the incidence of sporotrichosis as a zoonotic epidemic has increased, particularly in Rio de Janeiro. Aim: In this work, we decided to evaluate some T-cell phenotypes involved in the immune response. Materials & methods: We used flow cytometry to quantify TCD4+ and TCD8+ and Treg from immunocompetent and immunosuppressed mice infected with Sporothrix specieswith different levels of virulence and pathogenicity. Results: It was demonstrated the predominance of TCD4+ over the TCD8+ cells in both groups, inoculated with all the species, and percentages of Treg observed in infected immunocompetent mice. Conclusion: This regulatory phenotype can be associated with a protective immunity in the initial periods of infection.

Characterisation of the transcriptome and proteome of SARS-CoV-2 reveals a cell passage induced in-frame deletion of the furin-like cleavage site from the spike glycoprotein

BackgroundSARS-CoV-2 is a recently emerged respiratory pathogen that has significantly impacted global human health. We wanted to rapidly characterise the transcriptomic, proteomic and phosphoproteomic landscape of this novel coronavirus to provide a fundamental description of the virus’s genomic and proteomic potential.MethodsWe used direct RNA sequencing to determine the transcriptome of SARS-CoV-2 grown in Vero E6 cells which is widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. Allied to this, we used tandem mass spectrometry to investigate the proteome and phosphoproteome of the same virally infected cells.ResultsOur integrated analysis revealed that the viral transcripts (i.e. subgenomic mRNAs) generally fitted the expected transcription model for coronaviruses. Importantly, a 24 nt in-frame deletion was detected in over half of the subgenomic mRNAs encoding the spike (S) glycoprotein and was predicted to remove a proposed furin cleavage site from the S glycoprotein. Tandem mass spectrometry identified over 500 viral peptides and 44 phosphopeptides in virus-infected cells, covering almost all proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein.ConclusionsDetection of an apparently viable deletion in the furin cleavage site of the S glycoprotein, a leading vaccine target, shows that this and other regions of SARS-CoV-2 proteins may readily mutate. The furin site directs cleavage of the S glycoprotein into functional subunits during virus entry or exit and likely contributes strongly to the pathogenesis and zoonosis of this virus. Our data emphasises that the viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.

Differential immunogene expression profile of European sea bass (Dicentrarchus labrax, L.) in response to highly and low virulent NNV

European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. Knowing the differences between the immune response against both kinds of isolates may be key to get more insight into the host mechanisms responsible for NNV virulence. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (Mut247+270Dl956, RGNNV virus displaying SJNNV-type amino acids at positions 247 and 270 of the capsid protein), presenting lower virulence. This study has been performed in brain and head kidney, and the main differences between the immunogene responses triggered by both viruses have been observed in brain. The immunogene response in this organ is stronger after inoculation with the most virulent virus, and the main differences involved genes related with IFN I system, inflammatory response, cell-mediated response, and apoptosis. The lower virulence of Mut247+270Dl956 to European sea bass can be associated with a delayed IFN I response, as well as an early and transitory inflammation and cell-mediated responses, suggesting that those can be pivotal elements in controlling the viral infection, and therefore, their functional activity could be analysed in future studies. In addition, this study supports the role of capsid amino acids at positions 247 and 270 as important determinants of RGNNV virulence to European sea bass.

Resistance genes mediate differential resistance to pine defensive substances \u03b1-Pinene and H2O2 in Bursaphelenchus xylophilus with different levels of virulence

The pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, is the pathogen of pine wilt disease (PWD) which can devastate forests. PWN can be of high or low severity and the mechanisms underlying the differences in virulence are unclear. Therefore, it is necessary to study the relationship between differentiation of PWN severity and its resistance to the main defensive substances of pine species (i.e., α-pinene and H2O2). The feeding rate and fecundity of PWN was examined at different levels of virulence under conditions of α-pinene and H2O2 stress. Moreover, the expression patterns of the main resistance genes of PWN with different virulence were determined under conditions of α-pinene and H2O2 stress. The feeding rate and fecundity of the high virulence strain AMA3 were higher than those of the low virulence strain YW4. The expression levels of the autophagy gene BxATG5, cytochrome P450 gene BxCYP33D3, and glutathione S-transferase genes BxGST1 and BxGST3 in AMA3 increased significantly upon exposure to α-pinene for 2 h, while these genes showed smaller degrees of upregulation in YW4. Under conditions of H2O2 stress, the expression levels of BxATG5, catalase genes Bxy-ctl-1and Bxy-ctl-2, and the 2-cysteine peroxiredoxin gene BxPrx in AMA3 were higher than those in YW4. These findings suggest that high virulence PWN has greater resistance to pine defensive substances α-pinene and H2O2 than low virulence PWN, and resistance genes mediate the differential resistance of PWN strains. This study will contribute to the clarification of the mechanism underlying virulence differentiation of PWN and will advance understanding of the pathogenic mechanism of PWD.

Haemosporidioses in wild Eurasian blackbirds (Turdus merula) and song thrushes (T. philomelos): an in situ hybridization study with emphasis on exo-erythrocytic parasite burden

BackgroundPasserine birds are frequently infected with diverse haemosporidian parasites. While infections are traditionally considered benign in wild birds, recent studies demonstrated mortalities of passerine species due to exo-erythrocytic development of the parasites, which can damage organs in affected hosts. However, exo-erythrocytic development remains insufficiently investigated for most haemosporidian species and thus little is known about the virulence of tissue stages in wild passerine birds. The aim of the present study was to investigate natural haemosporidian infections in deceased Eurasian blackbirds (Turdus merula) and song thrushes (Turdus philomelos) and to determine parasite burden and associated histological effects.MethodsFor molecular analysis, blood and tissue samples from 306 thrushes were screened for Plasmodium, Haemoproteus and Leucocytozoon parasites by nested PCR. For the detection of parasite stages in organ samples, tissue sections were subjected to chromogenic in situ hybridization (CISH) using genus- and species-specific probes targeting the rRNAs of parasites. Exo-erythrocytic parasite burden was semi-quantitatively assessed and histological lesions were evaluated in haematoxylin–eosin-stained sections.ResultsBy PCR, 179 of 277 Eurasian blackbirds and 15 of 29 song thrushes were positive for haemosporidians. Parasites of all three genera were detected, with Plasmodium matutinum LINN1 and Plasmodium vaughani SYAT05 showing the highest prevalence. CISH revealed significant differences in exo-erythrocytic parasite burden between lineages in Eurasian blackbirds, with P. matutinum LINN1 frequently causing high exo-erythrocytic parasite burdens in various organs that were associated with histological alterations. Song thrushes infected with P. matutinum LINN1 and birds infected with other haemosporidian lineages showed mostly low exo-erythrocytic parasite burdens. Two Eurasian blackbirds infected with Leucocytozoon sp. TUMER01 showed megalomeronts in various organs that were associated with inflammatory reactions and necroses.ConclusionThis study suggests that P. matutinum LINN1, a common lineage among native thrushes, regularly causes high exo-erythrocytic parasite burdens in Eurasian blackbirds, which may result in disease and mortalities, indicating its high pathogenic potential. The findings further illustrate that the same parasite lineage may show different levels of virulence in related bird species which should be considered when assessing the pathogenicity of haemosporidian parasite species. Finally, the study provides evidence of virulent Leucocytozoon sp. TUMER01 infections in two Eurasian blackbirds caused by megalomeront formation.

Stimulatory Effects of Boscalid on Virulence of Sclerotinia sclerotiorum Indicate Hormesis May Be Masked by Inhibitions.

Hormetic effects of fungicides on phytopathogens are of great importance for proper application of fungicides. The aim of the present study was to investigate the stimulatory effects of the fungicide boscalid on mycelial growth and virulence of the devastating plant pathogen Sclerotinia sclerotiorum. Boscalid in potato dextrose agar (PDA) at a dosage range from 0.0005 to 0.002 μg/ml exerted statistically significant (P ≤ 0.015) stimulations on mycelial growth of S. sclerotiorum, and the maximum stimulation magnitudes were 5.55 ± 0.73% (mean ± SD) for the four isolates tested. Boscalid in PDA at 0.02 μg/ml inhibited mycelial growth of isolates HLJ3H and HLJ4H by 15.0 and 8.9%, respectively. However, after the growth-inhibited mycelia were inoculated on rapeseed leaves, isolates HLJ3H and HLJ4H exhibited virulence stimulations of 8.7 and 17.8%, respectively, indicating that hormesis may be masked by inhibitions. Boscalid sprayed at 0.0001 to 0.1 μg/ml on detached rapeseed leaves had significant (P ≤ 0.041) stimulations on virulence of S. sclerotiorum, and the maximum stimulation magnitudes were 17.90 ± 5.94% (mean ± SD) for the four isolates tested. Experiments on 12 isolates with different levels of virulence showed there was a negative correlation (R = -0.663, P = 0.019) between the maximum virulence stimulation magnitude and virulence of S. sclerotiorum in the absence of fungicide. Boscalid at stimulatory concentrations had no significant effect on the expression levels of three virulence-associated genes that encode cutinase (SsCut), polygalacturonase (SsPG1), and oxaloacetate acetylhydrolase (SsOah1). The molecular mechanisms for hormetic effects of boscalid on S. sclerotiorum remain to be studied in the future.

Resistance of spring wheat cultivars to pathogens of leaf diseases and root rot in the Right-bank Forest Steppe of Ukraine

Goal. Determine the degree of resistance of spring wheat cultivars to pathogens and root rot in the Right-bank Forest Steppe of Ukraine and identify promising sources of resistance.
 Methods. Laboratory — selection of infection material with different levels of virulence, development of infection material of leaf rust and septoria of wheat; field — creation of artificial infection and provocative backgrounds of pathogens and assessment of resistance of the collection of cultivars to this phytopathogens; analytical in mathematical — analysis of the obtained data, calculation of the intensity of disease development.
 Results. In 2017—2019, 30 cultivars of spring wheat from the collections of the National Center for Plant Genetic Resources of Ukraine were provided by the Ustimovskaya Plant Research Station of the Institute of Plant Growing. V.Ya. Yuriev NAAS was evaluated for resistance to the main leaf pathogens: powdery mildew Blumeria graminis (DC.) E.O. Speer f. sp. tritici Em. Marchal, brown rust Puccinia recondita f. sp. tritici Rob. et Desm. (syn. Puccinia triticina), leaf septoria Zymoseptoria tritici Rob. et Desm., pyrenophora Pyrenophora tritici-repentis (Died.) Drechsler and root rot. The collection included cultivars of Ukrainian, Russian, Kazakh and Kyrgyz breeding. Assessment of resistance to powdery mildew, pyrenophora and root rot was performed on a natural infectious background, and the pathogens of septoria and leaf rust on the created artificial complex infectious background during the period of maximum disease development. Cultivars were characterized by their degree of stability using a 9 — point immunological scale. According to the research results, the degree of resistance of varieties of Ukrainian and foreign breeding to pathogens of leaf diseases and root rot was established. Among the studied spring wheat collection, a number of cultivars were identified that showed resistance both to individual pathogens and to a group of diseases in the Right-Bank Forest Steppe of Ukraine: to powdery mildew — 4, brown rust — 20, septoria — 8, pyrenophorosis — 8, roots — 7. The group resistance was characterized by 13 spring wheat cultivars.
 Conclusions. Valuable sources of resistance are cultivars of Oksamit Mironovsky (UKR) and Lebedushka (RUS) with resistance to the group of pathogens of powdery mildew, leaf rust and pyrenophorosis, characteristic of the Right-Bank Forest Steppe zone of Ukraine. Other spring wheat cultivars that have high resistance to both individual pathogens and their groups are recommended to be involved in the breeding process to create new promising spring wheat cultivars with high resistance rates.

Genetic improvement in tobacco crops. Molecular mechanisms and regulation

Diseases produced by phytopathogenic fungi are one of the main causes of crop loss since humans started growing plants. Until now, these diseases have been mainly controlled by the improvement of plant resistance by means of selection and crossbreeding programs, the use of agrochemical products and an accurate crop rotation. Nevertheless, these strategies are not enough to control diseases caused by fungi because of the necessary long periods of time to carry out the crossbreeding and backcrossing as well as the selection of commercial varieties, which is plenty of time for new fungi types with different levels of virulence to appear. On the other hand, the use of agrochemical products is expensive and these eventually lack efficacy as a result of the evolution of the pathogen, apart from being extremely dangerous to the ecosystem. Implementing modern techniques of plant biotechnology as well as genetic engineering makes possible the obtainment of plants that protect themselves against fungi or fungal groups in short time. Tobacco is studied as a model to understand the molecular mechanisms of transference and regulation, which could be applied to crops of high economic importance such as the potato

Resistance of Capsicum annuum genotypes against various isolates of Phytophthora capsici from Java, Indonesia

Abstract. Wartono, Wiyono S, Syukur M, Giyanto, Lestari P. 2019. Resistance of Capsicum annuum genotypes against various isolates of Phytophthora capsici from Java, Indonesia. Biodiversitas 20: 3723-3730. Phytophthora capsici Leonian is one of the pathogenic oomycetes that can reduce chilli production worldwide. In this study, Indonesian local chilli genotypes were evaluated for their resistance to P.capsici on chilli in Java, under controlled conditions. P. capsici used in this study consisted of four isolates derived from chilli plants in Java Island. The chilli genotypes tested were 38 local chilli (Capsicum annuum L.) originated from Indonesia and 3 introduced C. annuum. Inoculation was carried out by drenching 5 ml suspension of zoospore (1 x 104) on the growing medium suitable for the oomycetes around the plant. Disease symptom was measured at 21 days after inoculation. Salicylic acid and b-1.3-glucanase analyses were conducted to 7-week old plant with specific interval. Molecular characterization to complement disease assay was carried out using SCAR primers (OpD04,717-F/D04,717-R). Significant differences were found in genotypes, isolates, and interactions between chilli genotypes x P. capsici isolates. Four tested isolates had different levels of virulence ranging from moderate (CpnsCK1, KdrRM3, WnsbCk) to high (WnsbCK2). The chilli genotypes revealed high partial resistance to P. capsici, as demonstrated by six genotypes resistant to CpnsCK1, two genotypes to KdrRM3, and one genotype resistant to WnsbCK1. Resistance of one of chilli pepper, Ungara was positively associated with an increase of salicylic acid and b-1.3-glucanase. There is no association between the SCAR marker with resistance of chilli to P. capsici suggested these pathogens from Java are probably controlled by another locus which is not detected by this SCAR marker.

Capsid amino acids at positions 247 and 270 are involved in the virulence of betanodaviruses to European sea bass

European sea bass (Dicentrarchus labrax) is severely affected by nervous necrosis disease, caused by nervous necrosis virus (NNV). Two out of the four genotypes of this virus (red-spotted grouper nervous necrosis virus, RGNNV; and striped jack nervous necrosis virus, SJNNV) have been detected in sea bass, although showing different levels of virulence to this fish species. Thus, sea bass is highly susceptible to RGNNV, whereas outbreaks caused by SJNNV have not been reported in this fish species. The role of the capsid protein (Cp) amino acids 247 and 270 in the virulence of a RGNNV isolate to sea bass has been evaluated by the generation of recombinant RGNNV viruses harbouring SJNNV-type amino acids in the above mentioned positions (Mut247Dl965, Mut270Dl965 and Mut247 + 270Dl965). Viral in vitro and in vivo replication, virus virulence and fish immune response triggered by these viruses have been analysed. Mutated viruses replicated on E-11 cells, although showing some differences compared to the wild type virus, suggesting that the mutations can affect the viral cell recognition and entry. In vivo, fish mortality caused by mutated viruses was 75% lower, and viral replication in sea bass brain was altered compared to non-mutated virus. Regarding sea bass immune response, mutated viruses triggered a lower induction of IFN I system and inflammatory response-related genes. Furthermore, mutations caused changes in viral serological properties (especially the mutation in amino acid 270), inducing higher seroconversion and changing antigen recognition.

Detection and identification of Xanthomonas pathotypes associated with citrus diseases using comparative genomics and multiplex PCR.

BackgroundIn Citrus cultures, three species of Xanthomonas are known to cause distinct diseases. X. citri subsp. citri patothype A, X. fuscans subsp. aurantifolii pathotypes B and C, and X. alfalfae subsp. citrumelonis, are the causative agents of cancrosis A, B, C, and citrus bacterial spots, respectively. Although these species exhibit different levels of virulence and aggressiveness, only limited alternatives are currently available for proper and early detection of these diseases in the fields. The present study aimed to develop a new molecular diagnostic method based on genomic sequences derived from the four species of Xanthomonas.ResultsUsing comparative genomics approaches, primers were synthesized for the identification of the four causative agents of citrus diseases. These primers were validated for their specificity to their target DNA by both conventional and multiplex PCR. Upon evaluation, their sensitivity was found to be 0.02 ng/µl in vitro and 1.5 × 104 CFU ml−1 in infected leaves. Additionally, none of the primers were able to generate amplicons in 19 other genomes of Xanthomonas not associated with Citrus and one species of Xylella, the causal agent of citrus variegated chlorosis (CVC). This denotes strong specificity of the primers for the different species of Xanthomonas investigated in this study.ConclusionsWe demonstrated that these markers can be used as potential candidates for performing in vivo molecular diagnosis exclusively for citrus-associated Xanthomonas. The bioinformatics pipeline developed in this study to design specific genomic regions is capable of generating specific primers. It is freely available and can be utilized for any other model organism.

Tropism of Newcastle disease virus strains for chicken neurons, astrocytes, oligodendrocytes, and microglia

BackgroundNewcastle disease (ND), which is caused by infections of poultry species with virulent strains of Avian orthoavulavirus-1, also known as avian paramyxovirus 1 (APMV-1), and formerly known as Newcastle disease virus (NDV), may cause neurological signs and encephalitis. Neurological signs are often the only clinical signs observed in birds infected with neurotropic strains of NDV. Experimental infections have shown that the replication of virulent NDV (vNDV) strains is in the brain parenchyma and is possibly confined to neurons and ependymal cells. However, little information is available on the ability of vNDV strains to infect subset of glial cells (astrocytes, oligodendrocytes, and microglia). The objective of this study was to evaluate the ability of NDV strains of different levels of virulence to infect a subset of glial cells both in vitro and in vivo. Thus, neurons, astrocytes and oligodendrocytes from the brains of day-old White Leghorn chickens were harvested, cultured, and infected with both non-virulent (LaSota) and virulent, neurotropic (TxGB) NDV strains. To confirm these findings in vivo, the tropism of three vNDV strains with varying pathotypes (SA60 [viscerotropic], TxGB [neurotropic], and Tx450 [mesogenic]) was assessed in archived formalin-fixed material from day-old chicks inoculated intracerebrally.ResultsDouble immunofluorescence for NDV nucleoprotein and cellular markers showed that both strains infected at least 20% of each of the cell types (neurons, astrocytes, and oligodendrocytes). At 24 h post-inoculation, TxGB replicated significantly more than LaSota. Double immunofluorescence (DIFA) with markers for neurons, astrocytes, microglia, and NDV nucleoprotein detected the three strains in all three cell types at similar levels.ConclusionThese data indicate that similar to other paramyxoviruses, neurons and glial cells (astrocytes, oligodendrocytes, and microglia) are susceptible to vNDV infection, and suggest that factors other than cellular tropism are likely the major determinant of the neurotropic phenotype.

Immunization With a Live-Attenuated RH:ΔNPT1 Strain of Toxoplasma gondii Induces Strong Protective Immunity Against Toxoplasmosis in Mice.

Toxoplasmosis, one of the most important health-threatening diseases worldwide, is caused by Toxoplasma gondii, which infects a wide range of warm-blooded animals and humans, leading to enormous health and socioeconomic concerns. T. gondii can establish chronic infection to evade the immune response in hosts. Once a chronic infection has been established, the available treatments cannot efficiently control this stage of T. gondii efficiently. Moreover, the available treatments rely only on a few drugs, such as sulfapyridine and pyrimethamine, that tend to have severe side effects. Given these factors, vaccination has been considered to be the most efficient method to prevent and control this disease. However, there is currently lack of effective vaccine available for use to prevent toxoplasmosis apart form Toxovax®, the only available vaccine, which is used in sheep to prevent abortion. To address this problem, we knocked out the NPT1 gene of the type I T. gondii strain using the CRISPR-Cas9 system, constructed a live-attenuated vaccine and evaluated its protective efficacy in a mouse model. Immunization of mice with RH:ΔNPT1 induced a high level of Toxoplasma-specific IgG1, IgG2a and total IgG 42 days after immunization. There was a significant increase in the levels of cytokines in the splenocyte suspensions of RH:ΔNPT1-infected mice, and a mixed Th1/Th2 response was induced in the mice. Remarkably, after heterologous challenges with tachyzoites of the RH, PYS and Pru strains and cysts of the Pru strain by different infection routes, the immunized animals were protected from toxoplasmosis with a 100% survival rate, in both acute and chronic infection. In addition, compared with control mice, the Pru cyst load was clearly reduced in the brains of RH:ΔNPT1-infected immunization-mice. Our study demonstrated that the RH:ΔNPT1 strain was able to evoke strong anti-Toxoplasma immune responses and provide effective protection against parasite strains with different levels of virulence, suggesting that the RH:ΔNPT1 strain may represent a promising live-attenuated vaccine against toxoplasmosis, which is worthy of further evaluation in food-producing animals and in definitive feline host.

Symptomatic and asymptomatic cases of African swine fever in Tanzania.

African swine fever (ASF) is an acute, highly contagious and deadly viral haemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV). In ASF endemic countries, there are an increasing number of reports on circulating ASFV strains with different levels of virulence causing a broad range of clinical symptoms in susceptible animals. Tanzania, where ASFV is endemic since 2001, recorded several outbreaks including symptomatic and asymptomatic cases between 2015 and 2017. We collected 35 clinical samples from four outbreaks for diagnostic confirmation and sequenced the partial B646L (p72), the full E183L (p54) gene, the central variable region of the B602L gene and the intergenic region between the I73R and I329L genes to characterize molecularly the new ASFV isolates and analyse their relatedness with previously reported Tanzanian and foreign isolates. We detected ASFV in 21 samples, 15 from symptomatic and six from asymptomatic pigs. Phylogenetic analyses based on the partial p72 gene and the complete p54 (E183L) genes revealed that the ASFVs in samples from symptomatic pigs belonged to genotypes II and those in samples from asymptomatic pigs belonged to genotype IX. The CVR profiles of the p72 genotype II and genotype IX isolates differed between each other and from previously published Tanzanian sequences. The sequence analysis of the intergenic region between the I73R and I329L for the 2017 genotype II isolates showed the absence of one GGAATATATA motif in those isolates. This study showed the simultaneous circulation of two different ASFV genotypes with different levels of pathogenicity in Tanzania. Since the existence of sub-clinically infected pigs may contribute to the persistence of the virus, our findings suggest continuous surveillance and characterization of ASFV isolates in disease-endemic regions.