WGS- versus ORF5-Based Typing of PRRSV: A Belgian Case Study.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most widespread and economically devastating diseases in the swine industry. Typing circulating PRRSV strains by means of sequencing is crucial for developing adequate control strategies. Most genetic studies only target the highly variable open reading frame (ORF) 5, for which an extensive database is available. In this study, we performed whole-genome sequencing (WGS) on a collection of 124 PRRSV-1 positive serum samples that were collected over a 5-year period (2015-2019) in Belgium. Our results show that (nearly) complete PRRSV genomes can be obtained directly from serum samples with a high success rate. Analysis of the coding regions confirmed the exceptionally high genetic diversity, even among Belgian PRRSV-1 strains. To gain more insight into the added value of WGS, we performed phylogenetic cluster analyses on separate ORF datasets as well as on a single, concatenated dataset (CDS) containing all ORFs. A comparison between the CDS and ORF clustering schemes revealed numerous discrepancies. To explain these differences, we performed a large-scale recombination analysis, which allowed us to identify a large number of potential recombination events that were scattered across the genome. As PRRSV does not contain typical recombination hot-spots, typing PRRSV strains based on a single ORF is not recommended. Although the typing accuracy can be improved by including multiple regions, our results show that the full genetic diversity among PRRSV strains can only be captured by analysing (nearly) complete genomes. Finally, we also identified several vaccine-derived recombinant strains, which once more raises the question of the safety of these vaccines.

Implementation and evaluation of different eradication strategies for Brachyspira hyodysenteriae.

BACKGROUND: Brachyspira infections are causing major losses to the pig industry and lead to high antimicrobial use. Treatment of Brachyspira (B.) hyodysenteriae infections may be problematic due to the high level of antimicrobial resistance. The present study implemented and evaluated farm-specific eradication programmes for B. hyodysenteriae in 10 different infected pig farms in Belgium. RESULTS: Ten pig farms clinically infected with B. hyodysenteriae volunteered to implement a farm-specific eradication programme. The programme depended on the farm and management characteristics, antimicrobial susceptibility of the B. hyodysenteriae strain and the motivation of the farmer. Two farms practiced total depopulation, six farms partial depopulation and two farms antimicrobial medication without depopulation. In addition, all farms implemented biosecurity measures, and faeces samples were tested for the presence of B. hyodysenteriae at 6, 9 and 12 months after the start of the program. Single Brachyspira isolates from before and after the programme were typed using multilocus sequence typing (MLST).Eradication was successful in four farms. Two of them (farrow-to-finish and finishing herd) had applied total depopulation and respected a vacancy period of at least 3 weeks. A third farm (gilt farm) practised partial depopulation, the rooms remained empty for 28 days and changed the source of breeding gilts. The fourth farm practised partial depopulation, the stables remained empty for 3 weeks, and used antimicrobial medication. The eradication programme was not successful in six farms. Two of the latter farms only used medication without partial depopulation. Four farms practiced partial depopulation, one of them combined it with antimicrobial medication. The cleaning and disinfection procedures, rodent control, stand-empty period and/or other biosecurity measures in the six farms were not always implemented properly. In two of three farms, isolates belonging to the same MLST type were found before and after eradication. CONCLUSIONS: Total depopulation or partial depopulation combined with implementing strict biosecurity measures allowed eradication of B. hyodysenteriae from clinically infected pig farms. Programmes based on antimicrobials without depopulation or partial depopulation without strictly adhering to all suggested biosecurity measures were not successful. Stockmanship and motivation of the farmer to permanently maintain high biosecurity standards are essential for success.

Presence and mechanisms of acquired antimicrobial resistance in Belgian Brachyspira hyodysenteriae isolates belonging to different clonal complexes.

Swine dysentery (SD) is an economically important disease for which antimicrobial treatment still occupies an important place to control outbreaks. However, acquired antimicrobial resistance is increasingly observed in Brachyspira hyodysenteriae. In this study, the Minimal Inhibitory Concentrations (MIC) of six antimicrobial compounds for 30 recent Belgian B. hyodysenteriae isolates were determined using a broth microdilution method. In addition, relevant regions of the 16S rRNA, 23S rRNA and the L3 protein encoding genes were sequenced to reveal mutations associated with acquired resistance. Finally, a phylogeny was reconstructed using minimal spanning tree analysis of multi locus sequence typing of the isolates. For lincomycin, doxycycline, tylosin and tylvalosin, at least 70% of the isolates did not belong to the wild-type population and were considered to have acquired resistance. For valnemulin and tiamulin, this was over 50%. In all isolates with acquired resistance to doxycycline, the G1058C mutation was present in their 16S rRNA gene. All isolates showing acquired resistance to lincomycin and both macrolides displayed the A2058T mutation in their 23S rRNA gene. Other mutations in this gene and the N148S mutation in the L3 protein were present in both wild-type isolates and isolates considered to have acquired resistance. Multi locus sequence analysis revealed a previously undescribed clonal complex, with 4 novel sequence types in which the majority of isolates showed acquired resistance to all tested antimicrobial products. In conclusion, acquired antimicrobial resistance is widespread among Belgian B. hyodysenteriae isolates. The emergence of multi-resistant clonal complexes can pose a threat to swine industry.