Ticks are one of the principal arthropod vectors of human and animal infectious diseases. Whereas the prevalence of tick-borne encephalitis virus in ticks in Europe is well studied, there is less information available on the prevalence of the other tick-borne viruses (TBVs) existing worldwide. The aim of this study was to improve the epidemiological survey tools of TBVs by the development of an efficient high-throughput test to screen a wide range of viruses in ticks.In this study, we developed a new high-throughput virus-detection assay based on parallel real-time PCRs on a microfluidic system, and used it to perform a large scale epidemiological survey screening for the presence of 21 TBVs in 18,135 nymphs of I. ricinus collected from five European countries. This extensive investigation has (i) evaluated the prevalence of four viruses present in the collected ticks, (ii) allowed the identification of viruses in regions where they were previously undetected.In conclusion, we have demonstrated the capabilities of this new screening method that allows the detection of numerous TBVs in a large number of ticks. This tool represents a powerful and rapid system for TBVs surveillance in Europe and could be easily customized to assess viral emergence.

Streptococcus suis is a porcine pathogen, causing severe invasive infections. S. suis serotype 9 is increasingly causing disease in Dutch and Chinese pig herds, but it is unknown whether all serotype 9 isolates are equally virulent and markers that can identify virulent strains are not available. Therefore, discrimination between virulent isolates and carriage isolates typically not associated with disease, is currently not possible. We collected tonsillar S. suis isolates from 6 herds not previously diagnosed with S. suis infections, and clinical S. suis isolates of previously diseased pigs. We confirmed the virulence of a virulent type strain and one representative clinical isolate, and the lack of virulence of two carriage isolates, in a pig infection model. Phylogenetic analysis of whole genome sequences of 124 isolates resulted in 10 groups, of which two were almost uniquely populated by clinical isolates. The population structure of S. suis serotype 9 appears highly diverse. However, analysis of the capsule loci sequences showed variation in a single region which fully correlated with a virulent genotype. Transmission electron microscopy suggested differences in capsule thickness between carriage and clinical genotypes. In conclusion, we found that that the S. suis serotype 9 population in the Netherlands is diverse. A distinct virulence-associated lineage was identified and could be discriminated based on the capsule locus sequence. Whilst the difference in virulence cannot be directly attributed to the DNA sequence, the correlation of capsule locus sequence with virulence could be used in the development of diagnostic tests to identify potential virulent S. suis serotype 9 in pigs.

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We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [gdh], and the gene encoding the extracellular protein factor [epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population.

The available differentiating tests for Chlamydia are based on detection of genetic material and only give information about the actual infection status, but reveal nothing of past infections. As the use of serological methods increases the window of detection, the goal of this study was to investigate if it is possible to develop a differentiating serological test for antibodies against Chlamydia species in chicken sera. Focus was on C. psittaci, C. gallinacea, and two closely related species, i.e. C. abortus and C. avium. To enable differentiating serology, a bead-based Luminex suspension array was constructed, using peptides as antigens, derived from known immunoreactive Chlamydia proteins. For the majority of these peptides, species-specific seroreactivity in mammalian sera has been reported in literature. The suspension array correctly identified antibodies against various Chlamydia species in sera from experimentally infected mice, and was also able to differentiate between antibodies against C. psittaci and C. gallinacea in sera from experimentally infected chickens. In field sera, signals were difficult to interpret as insufficient sera from experimentally infected chickens were available for evaluating the seroreactivity of all peptides. Nevertheless, results of the suspension array with field sera are supported by published data on the occurrence of C. gallinacea in Dutch layers, thereby demonstrating the proof of concept of multiplex serology for Chlamydial species in poultry.