Betaarterivirus suid 1 and 2 are the causative agents of porcine reproductive and respiratory syndrome (PRRS), which is one of the most significant diseases of the swine industry, causing significant economic losses in the main pig producing countries. Here, we report the development of a novel, RNA-based in situ hybridization technique (RNAscope) to detect PRRS virus (PRRSV) RNA in lung tissues of experimentally infected animals. The technique was applied to lung tissues of 20 piglets, which had been inoculated with a wild-type, highly pathogenic PRRSV-1 strain. To determine the RNAscope’s applicability as a semi-quantitative method, we analysed the association between the proportion of the virus-infected cells measured with an image analysis software (QuPath) and the outcome of the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) tests performed in parallel. The results of the quantitative approach of these two molecular biological methods show significant association (pseudo R2 = 0.3894, p = 0.004). This is the first time RNAscope assay has been implemented for the detection of PRRSV-1 in experimental animals.

Reproductive disorders caused by porcine reproductive and respiratory syndrome virus-1 are not yet fully characterized. We report QuPath-based digital image analysis to count inflammatory cells in 141 routinely, and 35 CD163 immunohistochemically stained endometrial slides of vaccinated or unvaccinated pregnant gilts inoculated with a high or low virulent PRRSV-1 strain. To illustrate the superior statistical feasibility of the numerical data determined by digital cell counting, we defined the association between the number of these cells and endometrial, placental, and fetal features. There was strong concordance between the two manual scorers. Distributions of total cell counts and endometrial and placental qPCR results differed significantly between examiner1’s endometritis grades. Total counts’ distribution differed significantly between groups, except for the two unvaccinated. Higher vasculitis scores were associated with higher endometritis scores, and higher total cell counts were expected with high vasculitis/endometritis scores. Cell number thresholds of endometritis grades were determined. A significant correlation between fetal weights and total counts was shown in unvaccinated groups, and a significant positive correlation was found between these counts and endometrial qPCR results. We revealed significant negative correlations between CD163+ counts and qPCR results of the unvaccinated group infected with the highly virulent strain. Digital image analysis was efficiently applied to assess endometrial inflammation objectively.

Background: Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease with widespread distribution and is currently a major cause of economic loss in pig production. In Hungary, a National PRRS Eradication Program was introduced in order to reach a more efficient, economic and competitive international market position. The EU also approved the program, but the resultant legal obligations placed a burden on Hungarian producers in order to comply with EU competition rules. The veterinary authorities carried out the implementation of the program with the consent, continuous support and monitoring by the organizations of the pig industry as well as a scientific committee. The PRRS eradication program in Hungary was based on a regional territorial principle and was obligatory for every swine farm within those regions. In Hungary large fattening farms operate as all-in/all-out or continuous flow system. The large-scale breeding herds are predominantly farrow-to-finish types. Although its significance has decreased in recent decades, 20% of the Hungarian pig population is still kept in small-scale (backyard) farms (< 100 animals). Each PRRS-infected large-scale farm had to develop a unit-adapted eradication plan, including external and internal biosecurity measures, vaccinations, etc. It was crucial to render each fattening unit free of the disease since fattening units play a significant role in spreading the virus across the country. Eradication efforts mainly implemented depopulation-repopulation methods, with some farms using the test and removal method instead. Results: As the eradication progressed over the years, the introduction of infected fattening pigs was restricted. Because of these measures, by the end of 2018, Hungarian large-scale fattening farms became free of PRRS. The PRRS-free status of the small-scale herds was achieved by the end of 2015, and this status was maintained from 2016–2021. By the 31st of December in 2021, all breeding pigs at large scale-farms in Hungary had become free of wild type PRRS virus. By the 31st of March in 2022, the total pig population of the country, including all backyard farms and fattening units, had achieved PRRS-free status. Conclusions: The future goal is to secure and maintain the PRRS free status of Hungary by strict import regulations of live animals combined with continuous and thorough screening of incoming and resident herds for the presence of the virus.