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Background A pregnant gilt infected with porcine reproductive and respiratory syndrome virus (PRRSV) can transmit the virus to her fetuses across the maternal-fetal-interface resulting in varying disease outcomes. However, the mechanisms leading to variation in fetal outcome in response to PRRSV infection are not fully understood. Our objective was to assess targeted immune-related gene expression patterns and pathways in the placenta and fetal thymus to elucidate the molecular mechanisms involved in the resistance/tolerance and susceptibility of fetuses to PRRSV2 infection. Fetuses were grouped by preservation status and PRRS viral load (VL): mock infected control (CTRL), no virus detected (UNINF), virus detected in the placenta only with viable (PLCO-VIA) or meconium-stained fetus (PLCO-MEC), low VL with viable (LVL-VIA) or meconium-stained fetus (LVL-MEC), and high VL with viable (HVL-VIA) or meconium-stained fetus (HVL-MEC). Results The host immune response was initiated only in fetuses with detectable levels of PRRSV. No differentially expressed genes (DEG) in either the placenta or thymus were identified in UNINF, PLCO-VIA, and PLCO-MEC when compared to CTRL fetuses. Upon fetal infection, a set of core responsive IFN-inducible genes (CXCL10, IFIH1, IFIT1, IFIT3, ISG15, and MX1) were strongly upregulated in both tissues. Gene expression in the thymus is a better differentiator of fetal VL; the strong downregulation of several innate and adaptive immune pathways (e.g., B Cell Development) are indicative of HVL. Gene expression in the placenta may be a better differentiator of fetal demise than the thymus, based-on principle component analysis clustering, gene expression patterns, and dysregulation of the Apoptosis and Ubiquitination pathways. Conclusion Our data supports the concept that fetal outcome in response to PRRSV2 infection is determined by fetal, and more significantly placental response, which is initiated only after fetal infection. This conceptual model represents a significant step forward in understanding the mechanisms underpinning fetal susceptibility to the virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe reproductive failure characterized by high fetal morbidity and mortality leading to substantial economic losses to the swine industry. Evaluation of spatiotemporal transmission of PRRSV at the maternal-fetal interface (MFI) is critical for understanding fetal infection. Localization of PRRSV-2 strain NVSL 97-7895 at different regions of the MFI in 20 pregnant gilts at 2, 5, 8, 12 and 14 days post inoculation (dpi) were analyzed by immunofluorescence (IF). Samples of MFI were collected from 15 inoculated and 5 control gilts and transplacental PRRSV transmission assessed in randomly selected fetuses from each litter. Localization of NVSL 97-7895 antigen immunoreactivity in the MFI was focused in three major areas: endometrial connective tissues (ENDO), the feto-maternal junction (FMJ) and fetal placenta (PLC). NVSL 97-7895 was detected at the FMJ by 2 dpi. At 2, 5 and 8 dpi, NVSL 97-7895 was localized within the ENDO and FMJ, whereas at 12 and 14 dpi, it was mainly localized in the PLC. Using a novel IF strategy for counting and size sorting NVSL 97-7895 viral antigen in situ, results of this study indicate that non-cell associated mechanisms are involved in PRRSV transmission across the MFI.

Background Mechanisms of fetal death following maternal PRRSV2 infection remain uncharacterized, although hypoxia from umbilical cord lesions and/or placental detachment due to apoptosis are hypothesized. We performed two experiments examining hypoxia and apoptosis in PRRSV-infected and non-infected, third-trimester fetuses to elucidate possible associations with fetal death. Fetuses were selected based on four phenotypic infection groups: fetuses from non-challenged control gilts (CTRL); low viral load fetuses (LVL; Exp 1) or uninfected fetuses (UNINF; Exp 2) from inoculated gilts; viable high viral load fetuses (HVL-VIA); and HVL meconium-stained fetuses (HVL-MEC). Results In experiment 1, paraffin embedded fetal tissues collected 21 days post maternal infection (DPI) were examined for DNA fragmentation associated with apoptosis. Positively stained foci were larger and more numerous (P < 0.05) in heart, liver, and thymus of HVL-VIA and HVL-MEC compared to CTRL and LVL fetuses. In experiment 2, group differences in gene expression within the hypoxia (HIF1a, IDO1, VEGFa, LDHA, NOS2, NOX1) and apoptosis (CASP3, CASP7, CASP8, CASP9, RIPK1, RIPK3) pathways were assessed by RT-qPCR in fetal tissues collected at 12 DPI. High viral load fetuses showed differential expression relative to the CTRL and UNINF (P < 0.05 for all). Brain tissue from HVL-VIA and HVL-MEC fetuses presented increased expression of CASP7, CASP8, RIPK3, HIF1a and IDO1. Fetal heart showed increased expression of CASP8, HIF1a, IDO and NOX1 and a decrease in NOS2 expression in infected groups. CASP7, CASP9, RIPK1 and RIPK3 were only increased in the heart of HVL-VIA while VEGFa was only increased for HVL-MEC fetuses. Thymus from HVL-MEC had decreased expression of CASP9 and there was increased IDO1 in all infected fetuses. Conclusions There is strong evidence of apoptosis occurring in the heart, liver and thymus of highly viral load fetuses at 21 DPI. Furthermore, there was clear upregulation of apoptotic genes in the heart of high viral load infected fetuses and less prominent upregulation in the brain of PRRSV-infected fetuses, whereas thymus appears to be spared at 12 DPI. There was no strong evidence of hypoxia at 12 DPI in brain and thymus but some indication of hypoxia occurring in fetal heart.

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PRRSV infection in third-trimester pregnant sows can lead to fetal death and abortions, although the mechanisms triggering these effects are not well understood. Since resistant and susceptible fetuses can coexist in the same litter, we propose that there may be differential mechanisms used by some fetuses to evade infection and/or disease progression. Our objectives were to investigate possible differences in the metabolome of PRRSV-infected and non-infected fetuses, as well as the interaction of altered intrauterine growth development and PRRSV infection to elucidate possible causes of fetal death following PRRSV infection. Near-term serum samples collected from fetuses on gestation day 106, 21 days post PRRSV-2 infection, were processed by direct flow injection mass spectrometry (DI-MS) and nuclear magnetic resonance (NMR) techniques. Experiment one investigated disease progression with 24 fetuses selected from each of four phenotypic groups: fetuses from non-inoculated gilts (CTRL); fetuses from inoculated gilts that escaped infection (UNINF); infected high viral load viable fetuses (INF); and infected high viral load meconium-stained fetuses (MEC). Experiment two investigated the interaction of intrauterine growth retardation (IUGR) and PRRSV infection by analyzing differences among: non-infected normal development (CON-N); CON-IUGR; PRRS infected normal development (PRRS-N); and PRRS-IUGR. Univariate and multivariate (PCA, PLS-DA) statistics determined group differences among various contrasts, and the most important metabolites associated with disease progression and fetal development. Significant differences in the metabolome were observed, especially between PRRSV-negative fetuses (CTRL and UNINF) and MEC fetuses, while INF fetuses appear to span both groups. The two metabolites with highest variable importance in projection (VIP) scores related to disease progression were alpha-aminoadipic acid (alpha-AAA) and kynurenine (KYN), having the highest concentration in MEC and INF fetuses, respectively, compared to CTRL and UNINF. In experiment two, non-IUGR fetuses were found to have increased levels of lysoPCs, PCs and amino acids compared to IUGR fetuses, while the near complete absence of lysoPCs and PCs in IUGR fetuses, even during infection, indicate a distinctive response to infection compared to non-growth retarded fetuses. Possible markers of PRRSV fetal susceptibility, such as alpha-AAA, kynurenine and lysoPCs, are presented and discussed.

Background: Staphylococcus (S.) aureus is an important nosocomial pathogen in humans and animals worldwide. The commonest class of antibiotics used to treat staphylococcal infections is the β-lactams. Frequently, S. aureus strains show high resistance to methicillin and other β-lactam antibiotics, called “Methicillin-resistant Staphylococcus aureus” (MRSA). Although MRSA has emerged at slower rate in domestic animals, it has frequently been found in the nasal cavity of healthy piglets and its transmission between pigs and swine handlers has already been studied. The aim of this work was to assess the presence of MRSA in finishing pigs in the state of Rio Grande do Sul, Brazil.Materials, Methods & Results: A total of 350 nasal swabs were collected from 10 to 20 week old finishing pigs. Sampling was performed in five pig farms in northeast Rio Grande do Sul State, Brazil. Swabs were stored in tubes without transport medium and carried to the laboratory under refrigeration. The specimens were cultured in selective and differential Agar (Baird Parker) and then were incubated at 37ºC for 48 h. After isolation of typical colonies of S. aureus, they were inoculated in BHI (Brain Heart Infusion) broth at 37ºC for 24 h and tested for tube coagulase activity. Coagulase positive samples were selected for growth in Oxacillin Resistant Screening Agar (ORSA) supplemented with 2 mg/L of oxacillin. This media contains aniline blue to demonstrate mannitol fermentation. Oxacillin and 5.5% NaCl have the capacity to reduce the growth of non-staphylococcal bacteria, selecting for MRSA.  Blue colonies growth after 24 to 48 h of incubation at 37ºC indicate the presence of positive MRSA strains. Specimens with at least one colony growing in ORSA within 48 h were considered resistant. Linear regression was performed in order to identify the association between herd size and MRSA frequency (SAS 9.4, 2012). Growth of S. aureus occurred on 18.0% of the samples and differences among farms were found. However, after incubation in ORSA only 18 (5.1%) were MRSA positive, ranging from zero to 12.5% among farms.  Significant correlation between herd size and MRSA frequency (adjusted r² = 0.978; P = 0.001) was observed.Discussion: In a previous study in pig herds in Brazil examining swine nasal swabs, 22.5% was positive for S. aureus and none for MRSA. One of 5 farms tested in our work also had no positive animals and 4 of them showed low frequencies, ranging from 1.7% to 12.5% with an average of 5.1%. Our results were similar to those found in Asian countries, but were very different from European data. Some factors can be associated with MRSA frequency in pig farms, such as dust, air contamination, poor hygiene, age, herd size, replacement rate and number of sources. In the present work we found a strong positive correlation (r² adjusted = 0.978; P = 0.001) between herd size and MRSA frequency, such as detected by previous authors. It might occur due to a higher risk of bacterial introduction and higher pressure of infection, easing dissemination of MRSA. Herd size may be a crucial factor to explain the frequencies found, since farms had very similar facilities and handling practices. Although the average frequency has been low in this work when compared to other countries, MRSA was present in almost all farms. This bacteria is able to transmit mecA gene to S. aureus susceptible populations, increasing MRSA frequencies over time.

Porcine reproductive and respiratory syndrome virus (PRRSV) is transmitted vertically, causing fetal death in late gestation. Spatiotemporal distribution of virus at the maternal–fetal interface (MFI) is variable, and accurate assessment of viral concentration and lesions is thus subject to sampling error. Our objectives were: 1) to assess whether viral load and lesion severity in a single sample of endometrium (END) and placenta (PLC), collected near the base of the umbilical cord (the current standard), are representative of the entire organ; and 2) to compare sampling strategies and evaluate if spatial variation in viral load can be overcome by pooling of like-tissues. Spatially distinct pieces of END and PLC of 24 fetuses from PRRSV-2–infected dams were collected. PRRSV RNA quantified by RT-qPCR was compared in 5 individual pieces per fetus and in respective pools of tissue and extracted RNA. Three distinct pieces of MFI were assessed for histologic severity. Concordance correlation and kappa inter-rater agreement were used to characterize agreement among individual samples and pools. The viral load of individual samples and pools of END had greater concordance to a referent standard than did samples of PLC. Larger pool sizes had greater concordance than smaller pool sizes. Average viral load and lesion severity did not differ by location sampled, and no technical advantages of pooling tissues versus RNA extracts were found. We conclude that multiple pieces of MFI tissues must be evaluated to accurately assess lesion severity and viral load. Three pieces per fetus provided a reasonable balance of cost and logistic feasibility.