Novel clades of tick-borne pathogenic nairoviruses in Europe.

Members of the Orthonairovirus genus (family Nairoviridae) include many tick-borne viruses of significant human and animal health impact, with several recently-documented pathogenic viruses lacking sufficient epidemiological information. We screened 215 adult ticks of seven species collected in Bulgaria, Georgia, Latvia and Poland for orthonairoviruses, followed by nanopore sequencing (NS) for genome characterization. Initial generic amplification revealed Sulina virus (SULV, Orthonairovirus sulinaense), for which an updated amplification assay was used, revealing an overall prevalence of 2.7% in Ixodes ricinus ticks from Latvia. Three complete and additional partial SULV genomes were generated, that consistently formed a separate, distinct clade with further intragroup divergence in the maximum likelihood analyses. Comparisons with previously described viruses from Romania exhibited similar genome topologies, albeit with divergent motifs and cleavage sites on the glycoprotein precursor. Preliminary evidence of recombination involving the S segment was documented, in addition to variations in predicted viral glycoproteins. Generic screening further identified Tacheng tick virus 1 (TCTV1, Orthonairovirus tachengense), with documented human infections, in Dermacentor reticulatus ticks from Poland, with a prevalence of 0.9%. Subsequent NS and assembly provided the first complete TCTV1 genome outside of China, where it was originally described. Phylogenetic analysis of virus genome segments revealed TCTV1-Poland as a discrete taxon within the TCTV1 cluster in the Orthonairovirus genus, representing a geographically segregated clade. Comparable genome topology with TCTV1 from China was observed, aside from minor variations in the M segment. Similar to SULV, TCTV1 exhibited several mismatches on previously described screening primer binding sites, likely to prevent amplification. These findings indicate presence of novel TCTV1 and SULV clades in Eastern Europe, confirming the expansion of orthonairoviruses with pathogenic potential.

Nanopore-based metagenomics reveal a new Rickettsia in Europe.

Accurate identification of tick-borne bacteria, including those associated with rickettsioses, pose significant challenges due to the polymicrobial and polyvectoral nature of the infections. We aimed to carry out a comparative evaluation of a non-targeted metagenomic approach by nanopore sequencing (NS) and commonly used PCR assays amplifying Rickettsia genes in field-collected ticks. The study included a total of 310 ticks, originating from Poland (44.2 %) and Bulgaria (55.8 %). Samples comprised 7 species, the majority of which were Ixodes ricinus (62.9 %), followed by Dermacentor reticulatus (21.2 %). Screening was carried out in 55 pools, using total nucleic acid extractions from individual ticks. NS and ompA/gltA PCRs identified Rickettsia species in 47.3 % and 54.5 % of the pools, respectively. The most frequently detected species were Rickettsia asiatica (27.2 %) and Rickettsia raoultii (21.8 %), followed by Rickettsia monacensis (3.6 %), Rickettsia helvetica (1.8 %), Rickettsia massiliae (1.8 %) and Rickettsia tillamookensis (1.8 %). Phylogeny construction on mutS, uvrD, argS and virB4 sequences and a follow-up deep sequencing further supported R. asiatica identification, documented in Europe for the first time. NS further enabled detection of Anaplasma phagocytophilum (9.1 %), Coxiella burnetii (5.4 %) and Neoehrlichia mikurensis (1.8 %), as well as various endosymbionts of Rickettsia and Coxiella. Co-detection of multiple rickettsial and non-rickettsial bacteria were observed in 16.4 % of the pools with chromosome and plasmid-based contigs. In conclusion, non-targeted metagenomic sequencing was documented as a robust strategy capable of providing a broader view of the tick-borne bacterial pathogen spectrum.

The expanding range of emerging tick-borne viruses in Eastern Europe and the Black Sea Region.

We analysed both pooled and individual tick samples collected from four countries in Eastern Europe and the Black Sea region, using metagenome-based nanopore sequencing (NS) and targeted amplification. Initially, 1337 ticks, belonging to 11 species, were screened in 217 pools. Viruses (21 taxa) and human pathogens were detected in 46.5% and 7.3%, respectively. Tick-borne viral pathogens comprised Tacheng Tick Virus 2 (TTV2, 5.9%), Jingmen Tick Virus (JMTV, 0.9%) and Tacheng Tick Virus 1 (TTV1, 0.4%). An association of tick species with individual virus taxa was observed, with the exception of TTV2, which was observed in both Dermacentor and Haemaphysalis species. Individual ticks from pools with pathogen detection were then further screened by targeted amplification and then NS, which provided extensive genome data and revealed probable pathogen Haseki Tick Virus (HTV, 10.2%). Two distinct TTV2 clades were observed in phylogenetic analysis, one of which included closely related Dermacentor reticulatus Uukuviruses. JMTV detection indicated integrated virus sequences. Overall, we observed an expansion of newly documented pathogenic tick-borne viruses into Europe, with TTV1 being identified on the continent for the first time. These viruses should be included in the diagnostic assessment of symptomatic cases associated with tick bites and vector surveillance efforts. NS is shown as a useful tool for monitoring tick-associated pathogens in pooled or individual samples.

Influence of Portland cement amendment on soil pH and residual soil termiticide performance.

Soil adjacent to new brick veneer work is likely to have a higher pH owing to the mixture of cement with the soil. In the Gainesville, FL, area, soil samples taken from such locations had a range of pH values from 9.0 to 10.1; similar soils used in bioassays had a pH of 5.6 before the addition of cement. Addition of 15 mg of Portland cement to 33 g of soil increased the pH to 6, and addition of 291 mg of Portland cement increased the pH to 9. The pH of soil amended with cement was stable for the first 5 months. After 10 months, soil pH values decreased from alkaline to near neutral in all cases. Eastern subterranean termite workers, Reticulitermes flavipes (Kollar), were exposed to the treated soil at pH 6-9 for 24 h, and percentage mortality was recorded at 5 days, 5 months and 10 months. Termite mortality significantly decreased at higher soil pHs for bifenthrin, chlorpyrifos, fipronil and imidacloprid treatments at 5 months and similarly for bifenthrin, permethrin, chlorpyrifos, fipronil and imidacloprid treatments at 10 months. There was an inverse linear relationship between soil pH and mortality. Increased soil pH diminished residual activity of termiticide in the following order: imidacloprid > fipronil > chlorpyrifos = bifenthrin > permethrin > cypermethrin.

Influence of soil compaction on tunnel network construction by the eastern subterranean termite (Isoptera: Rhinotermitidae).

Eastern subterranean termites, Reticulitermes flavipes (Kollar), workers were introduced into arenas containing low, moderate, and high compaction builder's sand (1.05 g/cm3, 1.18 g/cm3 or 1.35 g/cm3 bulk densities, respectively), and they immediately began tunneling. Termites built the tunnel network significantly fastest in soil of low compaction compared with moderately or highly compacted soil. In soil of low compaction, 221.67 +/- 4.73 cm of total tunnel distance was constructed in 1 d compared with only 96 cm of tunneling in highly compacted soil. At 14 d, total tunnel distance averaged 216.83 +/- 4.56 cm in soil of low compaction compared with 169.70 +/- 4.10 and 181.18 +/- 6.13 cm in moderately and highly compacted soil, respectively. Decreases in total tunnel distance between 1 and 14 d were caused by backfilling of seldom-used tunnels. Termites did the majority of tunneling during the first day of introduction into arenas. In soil of low and moderate compaction, termites essentially constructed the entire tunnel network within the first day, only modifying it by backfilling or maintaining tunnels. In highly compacted soil, 53% of the final tunnel network was constructed during the first day, 87% was constructed by the third day, and 97% was constructed by the seventh day. Soil compaction did not affect the number of primary tunnels or the number and diameter of secondary tunnels. The angle between the secondary tunnel and primary tunnel also was not significantly affected by soil compaction. However, the number of secondary tunnels in soil of low compaction (5.89 +/- 0.51) was significantly greater than in moderately (2.74 +/- 0.36) and highly (3.58 +/- 0.59) compacted soils.