Flanker: a tool for comparative genomics of gene flanking regions

Analysing the flanking sequences surrounding genes of interest is often highly relevant to understanding the role of mobile genetic elements (MGEs) in horizontal gene transfer, particular for antimicrobial-resistance genes. Here, we present Flanker, a Python package that performs alignment-free clustering of gene flanking sequences in a consistent format, allowing investigation of MGEs without prior knowledge of their structure. These clusters, known as ‘flank patterns’ (FPs), are based on Mash distances, allowing for easy comparison of similarity across sequences. Additionally, Flanker can be flexibly parameterized to fine-tune outputs by characterizing upstream and downstream regions separately, and investigating variable lengths of flanking sequence. We apply Flanker to two recent datasets describing plasmid-associated carriage of important carbapenemase genes (bla OXA-48 and bla KPC-2/3) and show that it successfully identifies distinct clusters of FPs, including both known and previously uncharacterized structural variants. For example, Flanker identified four Tn4401 profiles that could not be sufficiently characterized using TETyper or MobileElementFinder, demonstrating the utility of Flanker for flanking-gene characterization. Similarly, using a large (n=226) European isolate dataset, we confirm findings from a previous smaller study demonstrating association between Tn1999.2 and bla OXA-48 upregulation and demonstrate 17 FPs (compared to the 5 previously identified). More generally, the demonstration in this study that FPs are associated with geographical regions and antibiotic-susceptibility phenotypes suggests that they may be useful as epidemiological markers. Flanker is freely available under an MIT license at https://github.com/wtmatlock/flanker.

Resistance to Cucumber Green Mottle Mosaic Virus in Cucumis melo

Cucumber green mottle mosaic virus (CGMMV) is a severe threat to melon production worldwide. At present, there are no cultivars available on the market which show an effective resistance or tolerance to CGMMV infection; only wild Cucumis species were reported as resistant. Germplasm accessions of Cucumis melo, as well as C. anguria, C. ficifolius, C. myriocarpus and C. metuliferus, were mechanically infected with isolates belonging to the European and Asian strain of CGMMV and screened for resistance by scoring symptom severity and comparing the accumulation of virus by qRT-PCR. The wild species C. anguria and C. ficifolius showed no symptoms and did not accumulate CGGMV following inoculation, while C. metuliferus was highly susceptible to the isolates of both strains of CGMMV. The virus accumulated also in C. myriocarpus and the European isolate produced symptoms, but the Asian isolate did not. Thirty C. melo accessions were susceptible to CGMMV. An isolate-dependent expression of symptoms was observed in 16 melon accessions: they showed mild and severe symptoms at 14 and 21 days after inoculation with the European and Asian isolate, respectively. Freeman’s Cucumber showed few or no symptoms following inoculation with the isolate of either CGMMV strain. This particular accession also showed reduced virus accumulation, whereas most other tested germplasm accessions showed significantly higher viral loads and, therefore, may well be a candidate for breeding programs aiming to reduce the losses produced by CGMMV with resistant commercial melon cultivars.

Outbreak of Porcine Reproductive and Respiratory Syndrome Virus 1 in Taiwan

Porcine reproductive and respiratory syndrome (PRRS) causes significant economic losses in the swine industry worldwide. The PRRS virus (PRRSV) can be divided into two species, PRRSV 1 (European) and PRRSV 2 (North American). In Taiwan, PRRSV 2 isolates are dominant and cause respiratory symptoms in nursing pigs. From October to November 2018, in a pig herd in central Taiwan, pregnant sows had abortions and stillbirths, and piglets suffered from respiratory disorders. Laboratory tests identified the presence of PRRSV 1 in serum from sows and suckling piglets in this scenario. The complete genome of the identified PRRSV 1 strain was genetically closely related to that of a European PRRSV vaccine strain (98.2%). This local European isolate is designated as PRRSV/NPUST-2789-3W-2/TW/2018 (NPUST2789). This report is the first to indicate an outbreak in Taiwan of a PRRSV 1 strain that shares a common evolutionary ancestor with the European PRRSV vaccine strain.

Computational analysis of the Plasmodiophora brassicae genome: mitochondrial sequence description and metabolic pathway database design

Plasmodiophora brassicae is an obligate biotrophic pathogenic protist responsible for clubroot, a root gall disease of Brassicaceae species. In addition to the reference genome of the P. brassicae European e3 isolate and the draft genomes of Canadian or Chinese isolates, we present the genome of eH, a second European isolate. Refinement of the annotation of the eH genome led to the identification of the mitochondrial genome sequence, which was found to be bigger than that of Spongospora subterranea, another plant parasitic Plasmodiophorid phylogenetically related to P. brassicae. New pathways were also predicted, such as those for the synthesis of spermidine, a polyamine up-regulated in clubbed regions of roots. A P. brassicae pathway genome database was created to facilitate the functional study of metabolic pathways in transcriptomics approaches. These available tools can help in our understanding of the regulation of P. brassicae metabolism during infection and in response to diverse constraints.

Field evaluation of a live vaccine against porcine reproductive and respiratory syndrome in fattening pigs

In this study, a live vaccine based on an European isolate of porcine reproductive and respiratory syndrome (Porcilis PRRS, Intervet) virus has been tested in order to determine the protection of fattening pigs against the respiratory form of the syndrome under field conditions. Ten thousand pigs in an infected farm were vaccinated against PRRS virus at the age of 6 weeks and were compared to non-vaccinated pigs with respect to their health status, mortality, performance parameters (average daily gain, average daily feed intake, feed conversion ratio) and the presence of specific pathogens in their lungs. The results have shown that treated pigs became less frequently sick and have demonstrated reduced mortality compared to untreated ones. As compared to non-vaccinated animals, PRRSvaccinated pigs also performed in a better way with respect to feed conversion ratio (P<0.05) and average daily gain (P<0.05), while feed intake was similar for both groups (P>0.05). Bacteriological examinations of the lungs have revealed increased incidence of respiratory bacterial infection in untreated pigs compared to treated ones. Also a tendency for faster antibody response was detected in the vaccinées. Finally, no clinical side effects were observed after the administration of the vaccine. The results of the present study show that immunization with Porcilis PRRS (Intervet) does protect fattening pigs against the respiratory manifestations of PRRS.

Mitochondrial DNA from the eradicated European Plasmodium vivax and P. falciparum from 70-year-old slides from the Ebro Delta in Spain

Phylogenetic analysis of Plasmodium parasites has indicated that their modern-day distribution is a result of a series of human-mediated dispersals involving transport between Africa, Europe, America, and Asia. A major outstanding question is the phylogenetic affinity of the malaria causing parasites Plasmodium vivax and falciparum in historic southern Europe—where it was endemic until the mid-20th century, after which it was eradicated across the region. Resolving the identity of these parasites will be critical for answering several hypotheses on the malaria dispersal. Recently, a set of slides with blood stains of malaria-affected people from the Ebro Delta (Spain), dated between 1942 and 1944, have been found in a local medical collection. We extracted DNA from three slides, two of them stained with Giemsa (on which Plasmodium parasites could still be seen under the microscope) and another one consisting of dried blood spots. We generated the data using Illumina sequencing after using several strategies aimed at increasing the Plasmodium DNA yield: depletion of the human genomic (g)DNA content through hybridization with human gDNA baits, and capture-enrichment using gDNA derived from P. falciparum. Plasmodium mitochondrial genome sequences were subsequently reconstructed from the resulting data. Phylogenetic analysis of the eradicated European P. vivax mtDNA genome indicates that the European isolate is closely related to the most common present-day American haplotype and likely entered the American continent post-Columbian contact. Furthermore, the European P. falciparum mtDNA indicates a link with current Indian strains that is in agreement with historical accounts.

Draft Genome Sequence of a European Isolate of the Apple Canker Pathogen Neonectria ditissima

The Sordariomycetes fungus Neonectria ditissima is a major pathogen of apples, causing canker on trees and fruit spoilage. We report here the draft genome sequence of a European strain isolated from cankerous tissue.

Draft Genome Sequence of Erwinia billingiae OSU19-1, Isolated from a Pear Tree Canker

Plant-associated Erwinia include pathogenic and nonpathogenic species . We report the 5.6-Mb genome sequence of Erwinia billingiae OSU19-1, isolated from a canker on a pear tree inoculated with Erwinia amylovora . OSU19-1 and a closely related European isolate, E. billingiae Eb661 T , share many similarities including 40 kb of plasmid sequence.

Additional data on Iranian trichodorids (Triplonchida: Trichodoridae) and proposal for a new synonymy

Molecular data on several species of trichodorids occurring in Iran are provided. Live specimens of two species, Trichodorus arasbaranensis and T. gilanensis, were collected from their type localities. One population of T. orientalis was collected from Arasbaran forests in Eastern Azarbayjan province. The species Paratrichodorus porosus, occurring in Gilan province, and one European isolate of P. pachydermus were also included in phylogenetic analysis. An Iranian population of T. minzi, representing only the second population found of this rare species, was recovered from soil samples collected from grasslands of north-western Iran and is in morphological congruence with the original description of the species. Besides morphological study, molecular phylogenetic study was performed on newly reported species using partial 28S rDNA data, and the phylogenetic relations of all sequenced species as inferred from Bayesian analysis are discussed. According to the phylogenetic tree, two newly described species, T. golestanensis and T. iranicus, formed a clade and further studies revealed they have the same morphology and molecular sequences of 28S rDNA D2-D3. Accordingly, T. golestanensis is regarded as a junior synonym of T. iranicus according to the Principle of Priority.