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Finch trichomonosis in Europe is caused by a Trichomonas gallinae subtype A1 strain, considered to be clonal because lacking genetic heterogeneity in partial genotyping. The disease recently emerged and has been associated with a 66% reduction of the British breeding greenfinch ( Chloris chloris ) population. In contrast, in the Netherlands, where trichomonosis was detected in 2009, the breeding greenfinch population continued to grow in subsequent years. This study aimed to elucidate whether this discrepancy in population trends is because Trichomonas infection in Dutch greenfinches is associated with less severe disease, i.e., disease being less fatal. Therefore, it characterized and quantified trichomonosis in a convenience sample of greenfinches found dead and examined post-mortem between 2009 and 2017 and compared results to published data from Great Britain. Trichomonads were detected by cytology, histology, or culture in 95/101 greenfinches. The birds with trichomonads all had microscopic lesions in the upper digestive tract consistent with trichomonosis, indicating the trichomonads caused disease. The occurrence of significant lesions due to other causes was low. Some greenfinches with trichomonosis showed no macroscopic lesions. These birds showed significantly less ulceration of the mucosa and less extensive heterophil infiltration, but extent of macrophage infiltration and presence of bacteria was similar to that of birds with macroscopic lesions, and significant lesions due to other causes were equally rare. Therefore, trichomonosis was considered similarly fatal in both groups. The frequency of fatal trichomonosis in the Dutch greenfinches did not differ significantly from that reported from Great Britain. Partial genotyping of the ITS1-5,8S-ITS2 and Fe-hydrogenase regions of T. gallinae was performed to detect genetic heterogeneity, that could indicate the presence of other, possibly less virulent, strains. In 60/63 samples there was full alignment of sequences with the clonal strain of T. gallinae subtype A1. The remaining three samples had the same single synonymous nucleotide difference in the Fe-hydrogenase region; however, pathology is these three was identical to the others. Collectively, the results provide no clear evidence for less severe disease as explanation for the discrepancy in census data trends. We conclude that trichomonosis is a threat concealed in Dutch breeding greenfinch census data., (Copyright © 2019 Rijks, Laumen, Slaterus, Stahl, Gröne and Kik.)

Rabbit haemorrhagic disease virus (RHDV) type 2 (GI.2/RHDV2/b) is an emerging pathogen in wild rabbits and in domestic rabbits vaccinated against RHDV (GI.1). Here we report the genome sequence of a contemporary RHDV2 isolate from the Netherlands and investigate the immunogenicity of virus-like particles (VLPs) produced in insect cells. RHDV2 RNA was isolated from the liver of a naturally infected wild rabbit and the complete viral genome sequence was assembled from sequenced RT-PCR products. Phylogenetic analysis based on the VP60 capsid gene demonstrated that the RHDV2 NL2016 isolate clustered with other contemporary RHDV2 strains. The VP60 gene was cloned in a baculovirus expression vector to produce VLPs in Sf9 insect cells. Density-gradient purified RHDV2 VLPs were visualized by transmission electron microscopy as spherical particles of around 30 nm in diameter with a morphology resembling authentic RHDV. Immunization of rabbits with RHDV2 VLPs resulted in high production of serum antibodies against VP60, and the production of cytokines (IFN-γ and IL-4) was significantly elevated in the immunized rabbits compared to the control group. The results demonstrate that the recombinant RHDV2 VLPs are highly immunogenic and may find applications in serological detection assays and might be further developed as a vaccine candidate to protect domestic rabbits against RHDV2 infection., Competing Interests: The authors declare no conflict of interest.