The situation in Europe concerning honeybees has in recent years become increasingly aggravated with steady decline in populations and/or catastrophic winter losses. This has largely been attributed to the occurrence of a variety of known and “unknown”, emerging novel diseases. Previous studies have demonstrated that colonies often can harbour more than one pathogen, making identification of etiological agents with classical methods difficult. By employing an unbiased metagenomic approach, which allows the detection of both unexpected and previously unknown infectious agents, the detection of three viruses, Aphid Lethal Paralysis Virus (ALPV), Israel Acute Paralysis Virus (IAPV), and Lake Sinai Virus (LSV), in honeybees from Spain is reported in this article. The existence of a subgroup of ALPV with the ability to infect bees was only recently reported and this is the first identification of such a strain in Europe. Similarly, LSV appear to be a still unclassified group of viruses with unclear impact on colony health and these viruses have not previously been identified outside of the United States. Furthermore, our study also reveals that these bees carried a plant virus, Turnip Ringspot Virus (TuRSV), potentially serving as important vector organisms. Taken together, these results demonstrate the new possibilities opened up by high-throughput sequencing and metagenomic analysis to study emerging new diseases in domestic and wild animal populations, including honeybees.

Compared to routine diagnostics, screening for pathogens in outbreak situations, with or without intentional release, poses demands on the detection technology to not only indicate the presence of already known causative agents but also novel and unexpected pathogens. The metagenomic approach to detecting viral pathogens, using unbiased high-throughput sequencing (HTS), is a well-established methodology with a broad detection range and wide applicability on different sample matrices. To prepare a sample for HTS, the common presequencing steps include homogenization, enrichment, separation (eg, magnetic separation), and amplification. In this initial study, we explored the benefits and drawbacks of preprocessing by sequence-independent, single-primer amplification (SISPA) of nucleic acids by applying the methodology to artificial samples. More specifically, a synthetic metagenome was divided into 2 samples, 1 unamplified and 1 diluted, and amplified by SISPA. Subsequently, both samples were sequenced using the Ion Torrent Personal Genome Machine (PGM), and the resulting datasets were analyzed by using bioinformatics, short read mapping, de novo assembly, BLAST-based taxonomic classification, and visualization. The results indicate that even though SISPA introduces a strong amplification bias, which makes it unsuitable for whole-genome sequencing, it is still useful for detecting and identifying viruses.

An in-depth analysis was performed on Swedish broiler producers that had delivered chickens with Campylobacter to slaughter over several years, in order to identify possible transmission routes and formulate effective measures to prevent chickens being colonized with Campylobacter. Between 2017 and 2019, 626 samples were collected at farm level and Campylobacter was isolated from 133 (21.2%). All C. jejuni and C. coli isolated from these samples were whole-genome sequenced, together with isolates from the corresponding cecum samples at slaughter (n = 256). Core genome multi-locus sequence typing (cgMLST) analysis, using schemes consisting of 1140 and 529 genes for C. jejuni and C. coli, respectively, revealed that nearby cattle, contaminated drinking water, water ponds, transport crates, and parent flocks were potential reservoirs of Campylobacter. A novel feature compared with previous studies is that measures were implemented and tested during the work. These contributed to a nationwide decrease in Campylobacter-positive flocks from 15.4% in 2016 to 4.6% in 2019, which is the lowest ever rate in Sweden. To conclude, there are different sources and routes of Campylobacter transmission to chickens from different broiler producers, and individual measures must be taken by each producer to prevent Campylobacter colonization of chickens.

Neonatal porcine diarrhoea of uncertain aetiology has been reported from a number of European countries. The aim of the present study was to use viral metagenomics to examine a potential viral involvement in this diarrhoea and to describe the intestinal virome with focus on eukaryotic viruses. Samples from the distal jejunum of 50 diarrhoeic and 19 healthy piglets from 10 affected herds were analysed. The viral fraction of the samples was isolated and nucleic acids (RNA and DNA fractions) were subjected to sequence independent amplification. Samples from diarrhoeic piglets from the same herds were pooled whereas samples from healthy piglets were analysed individually. In total, 29 clinical samples, plus two negative controls and one positive control consisting of a mock metagenome were sequenced using the Ion Torrent platform. The resulting sequence data was subjected to taxonomic classification using Kraken, Diamond and HMMER. In the healthy specimens, eight different mammalian virus families were detected (Adenoviridae, Anelloviridae, Astroviridae, Caliciviridae, Circoviridae, Parvoviridae, Picornaviridae, and Reoviridae) compared to four in the pooled diarrhoeic samples (Anelloviridae, Circoviridae, Picornaviridae, and Reoviridae). It was not possible to associate a particular virus family with the investigated diarrhoea. In conclusion, this study does not support the hypothesis that the investigated diarrhoea was caused by known mammalian viruses. The results do, however, indicate that known mammalian viruses were present in the intestine as early as 24–48 hours after birth, indicating immediate infection post-partum or possibly transplacental infection.

Background: Parascaris univalensis a pathogenic parasite of foals and yearlings worldwide. In recent years,Parascarisspp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adultP. univalensworms afterin vitroexposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways.Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10⁻⁹ M, 10⁻¹¹ M, 10⁻¹³ M), pyrantel citrate (10⁻⁶ M, 10⁻⁸ M, 10⁻¹⁰ M), thiabendazole (10⁻⁵ M, 10⁻⁷ M, 10⁻⁹ M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system.Results: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ − 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunitlgc-37, which was upregulated only in worms exposed to 10⁻⁹ M of ivermectin.Conclusions: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described inP. univalensand future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.