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The hippocampus participates in spatial navigation and behavioral processes, displays molecular plasticity in response to environmental challenges, and can play a role in neuropsychiatric diseases. The combined effects of inflammatory prenatal and postnatal challenges can disrupt the hippocampal gene networks and regulatory mechanisms. Using a proven pig model of viral maternal immune activation (MIA) matched to controls and an RNA-sequencing approach, the hippocampal transcriptome was profiled on two-month-old female and male offspring assigned to fasting, mimetic viral, or saline treatments. More than 2600 genes presented single or combined effects (FDR-adjusted p-value < 0.05) of MIA, postnatal stress, or sex. Biological processes and pathways encompassing messenger cyclic adenosine 3′,5′-monophosphate (cAMP) signaling were enriched with genes including gastric inhibitory polypeptide receptor (GIPR) predominantly over-expressed in the MIA-exposed fasting males relative to groups that differed in sex, prenatal or postnatal challenge. While this pattern was amplified in fasting offspring, the postnatal inflammatory challenge appeared to cancel out the effects of the prenatal challenge. The transcription factors C-terminal binding protein 2 (CTBP2), RE1 silencing transcription factor (REST), signal transducer and activator of transcription 1 (STAT1), and SUZ12 polycomb repressive complex 2 subunit were over-represented among the genes impacted by the prenatal and postnatal factors studied. Our results indicate that one environmental challenge can influence the effect of another challenge on the hippocampal transcriptome. These findings can assist in the identification of molecular targets to ameliorate the effects of pre-and post-natal stressors on hippocampal-associated physiology and behavior.

The influence of proinflammatory challenges, such as maternal immune activation (MIA) or postnatal exposure to drugs of abuse, on brain molecular pathways has been reported. On the other hand, the simultaneous effects of MIA and drugs of abuse have been less studied and sometimes offered inconsistent results. The effects of morphine exposure on a pig model of viral-elicited MIA were characterized in the prefrontal cortex of males and females using RNA-sequencing and gene network analysis. Interacting and main effects of morphine, MIA, and sex were detected in approximately 2000 genes (false discovery rate-adjusted p-value < 0.05). Among the enriched molecular categories (false discovery rate-adjusted p-value < 0.05 and −1.5 > normalized enrichment score > 1.5) were the cell adhesion molecule pathways associated with inflammation and neuronal development and the long-term depression pathway associated with synaptic strength. Gene networks that integrate gene connectivity and expression profiles displayed the impact of morphine-by-MIA interaction effects on the pathways. The cell adhesion molecules and long-term depression networks presented an antagonistic effect between morphine and MIA. The differential expression between the double-challenged group and the baseline saline-treated Controls was less extreme than the individual challenges. The previous findings advance the knowledge about the effects of prenatal MIA and postnatal morphine exposure on the prefrontal cortex pathways.

The effects of the practice of weaning in pigs have been documented, and include changes in organ function and immune mechanisms. However, the effects of weaning following exposure to maternal infection during gestation are incompletely characterized. We hypothesize that the disruption of physiological processes triggered by weaning can be influenced by the exposure of the pig to maternal immune signals elicited by a challenge during gestation. To test our hypothesis, the concentrations of biochemical indicators and cytokines were measured in the blood from 72 female and male pigs distributed across two weaning groups (nursed or weaned), and two gestational challenge groups (gilts infected with porcine reproductive and respiratory virus or healthy controls). Weaning had a significant effect on the concentration of albumin among females born from healthy gilts (P < 0.01). The albumin concentrations were higher in weaned relative to nursed pigs from healthy gilts. This pattern may be associated with the transition to a higher protein diet, whereas no differences were observed among pigs from infected gilts. The interaction between gestation and weaning stresses had a significant effect on the concentration of bicarbonate. The highest levels of bicarbonate were detected among nursed pigs from infected gilts. The lowest levels of bicarbonate were detected among weaned pigs from health gilts, and this could indicate metabolic acidosis. An interaction effect (P < 0.02) was detected for pro-inflammatory interleukin 4. The highest concentrations of interleukin 4 were detected in weaned females from infected gilts, while the lowest levels were detected among weaned males from health gilts. The studied biomarker patterns indicate that the effect of weaning on blood indicators can be influenced by the exposure of the pigs to challenges during gestation. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

Simple Summary The present study of blood chemical, cytokine and hormone indicators in male and female pigs offered insights into the combined effects of maternal immune activation and weaning stress. Our results indicate that maternal immune activation may grant higher tolerance to anion gap and bilirubin disruptions triggered by weaning stress. Therefore, management practices aimed at minimizing the effects of weaning should consider the interacting effect of immune activation during development. Abstract Weaning stress can elicit changes in the metabolic, hormone and immune systems of pigs and interact with prolonged disruptions stemming from maternal immune activation (MIA) during gestation. The present study advances the characterization of the combined effects of weaning stress and MIA on blood chemistry, immune and hormone indicators that inform on the health of pigs. Three-week-old female and male offspring of control gilts or gilts infected with the porcine reproductive and respiratory syndrome virus were allocated to weaned or nursed groups. The anion gap and bilirubin profiles suggest that MIA enhances tolerance to the effects of weaning stress. Interleukin 1 beta and interleukin 2 were highest among weaned MIA females, and cortisol was higher among weaned relative to nursed pigs across sexes. Canonical discriminant analysis demonstrated that weaned and nursed pigs have distinct chemistry profiles, whereas MIA and control pigs have distinct cytokine profiles. The results from this study can guide management practices that recognize the effects of the interaction between MIA and weaning stress on the performance and health of pigs.

Changes at the molecular level capacitate the plasticity displayed by the brain in response to stress stimuli. Weaning stress can trigger molecular changes that influence the physiology of the offspring. Likewise, maternal immune activation (MIA) during gestation has been associated with behavior disorders and molecular changes in the amygdala of the offspring. This study advances the understanding of the effects of pre- and postnatal stressors in amygdala gene networks. The amygdala transcriptome was profiled on female and male pigs that were either exposed to viral-elicited MIA or not and were weaned or nursed. Overall, 111 genes presented interacting or independent effects of weaning, MIA, or sex (FDR-adjusted P-value

Inflammatory signals elicited in response to stressors during gestation can affect fetal development and can have prolonged postnatal effects in the offspring. The objectives of the present study were to assess the effects of viral infection during gestation on nursed piglets, and to investigate possible modulating effects of fish oil. A total of 36 pigs were studied, distributed across three gilt treatment groups and sexes within treatment group. One-third of the piglets served as reference, being born from gilts that were not exposed to viral infection and receiving a non-supplemented diet. Another third of the piglets were born from gilts that were challenged with porcine reproductive and respiratory virus during the last third of gestation and receiving a non-supplemented diet. The remaining piglets were born from gilts that were challenged with the virus and were supplemented with fish oil (2.5 g of docosahexaenoic acid and eicosapentaeonic acid). All gilts received diets that met nutritional requirements during gestation and lactation (~90% corn and soybean meal). The piglets remained with the gilts and nursed until 22 d of age at which time blood was drawn and the levels of metabolic parameters were measured. The immune challenge during gestation had a significant effect on the level of globulin (P < 0.02). The globulin levels were higher in female piglets from gilts infected during gestation relative to control gilts. The blood level of triglycerides was similar between piglets from reference gilts and viral-infected gilts supplemented with fish oil, and lower than in piglets from non-supplemented infected gilts. Our results demonstrate that fish oil supplementation can modulate effects of infection during gestation on the piglet. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

The prolonged and sex-dependent impact of maternal immune activation (MIA) on the molecular pathways of the amygdala, a brain region that influences social, cognitive, and sexually dimorphic behaviors, is only partially understood. To address this gap, the effect of MIA elicited by porcine reproductive and respiratory syndrome virus (PRRSV) infection during gestation on the amygdala transcriptome of pigs was studied. Gene expression levels were measured using RNA-Seq on the amygdala for 3-week-old female and male offspring from MIA and control groups. Among the 328 genes that exhibited an MIA-by-sex effect, the majority annotated to functional categories relevant to behavioral abnormalities, including neuroactive ligand-receptor pathways, glutamatergic functions, and neuropeptide systems. Genes in these categories included corticotropin releasing hormone receptor 2, glutamate metabotropic receptor 4, glycoprotein hormones, alpha polypeptide, parathyroid hormone 1 receptor, vasointestinal peptide receptor 2, neurotensin, proenkephalin, and gastrin releasing peptide. These genes and functional categories have been associated with MIA-related schizophrenia and autism spectrum behavior disorders. The transcript and network dysregulation uncovered in this study advances the understanding necessary to develop treatments that ameliorate the effects of neurodevelopmental disorders caused by gestational MIA exposure. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

Costs and productivity losses associated with porcine reproductive and respiratory syndrome virus (PRRSV) outbreaks impact the swine industry. The objective of this study was to evaluate the effect of maternal immune activation (MIA) from a PRRSV challenge during gestation on the piglet serum concentrations of albumin and globulin, indicators of inflammation or organ damage. Eighteen pregnant Camborough gilts were inoculated with either PRRSV or saline on gestational day 76. After farrowing 106 piglets remained with the gilts until postnatal day (PD) 21. One group of piglets was weaned and group-housed while another group remained with the gilts until PD22. At PD 60, a group of weaned piglets was fasted for one day while the rest was fed to meet nutritional requirements. Serum concentrations of globulin and albumin were measured on PD 22 and 61 and were analyzed using a linear mixed model including the fixed effects of gilt challenge, piglet treatment and sex and the random effect of gilt. On PD 22, albumin was higher in weaned relative to suckled piglets from saline-treated gilts (P < 0.0305). Globulin was higher in weaned relative to suckled piglets (P < 0.0014) and in piglets from PRRSV-treated relative to saline-treated gilts (P < 0.0201). The albumin/globulin ratio was higher in weaned piglets from saline relative to PRRSV-treated gilts (P < 0.0367). On PD 61, albumin was higher in fasted relative to fed piglets (P < 0.0072). Globulin was higher in males relative to females from PRRSV-treated gilts (P < 0.0326), and the albumin/globulin ratio was higher in fasted relative to fed piglets (P < 0.0043). Our results indicate a positive correlation between serum protein concentrations and MIA, weaning or fasting stress that may be indicative of inflammation, organ dysfunction or nutritional imbalance. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

Maternal immune activation (MIA) during gestation can affect the ability of the offspring to cope with stress such as weaning. Cortisol, a glucocorticoid released in response to stress, is an established indicator of stress. The purpose of this study was to evaluate the cortisol response to weaning stress of offspring exposed to MIA during gestation. Camborough gilts were inseminated at approximately 235 days of age. On gestational day 76, gilts (n = 11) were inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) intranasally whereas saline was intranasally administered to Control gilts (n = 7). On postnatal day (PD) 21 approximately half of the male and female piglets remained with sows (suckled) while the rest were weaned. Blood was collected from the piglets prior to weaning on PD21 and at PD22. ELISA was used to measure cortisol in the serum. The analysis of cortisol concentration tested for the fixed effects of gilt treatment (PRRSV or Control), piglet sex (female or male), management (suckled or weaned), day of sampling (PD21 or PD22), interactions, and for Normality assumptions. At PD22 female weaned piglets had a higher level of cortisol when compared to female suckled piglets in both Control (P < 0.05) and PRRSV (P < 0.05). Males exhibited a similar trend that did not reach significance. Cortisol concentrations were lower in male piglets from Control relative to PRRSV-treated gilts and the differences were more pronounced among the suckled group. The results suggest that MIA elevates basal serum cortisol in unstressed males. This study is supported by USDA NIFA AFRI, 2018-67015-27413.

The prolonged effects of maternal immune activation in response stressors during gestation on the offspring’s molecular pathways after birth are beginning to be understood. An association between maternal immune activation and neurodevelopmental and behavior disorders such as autism and schizophrenia spectrum disorders has been detected in long-term gene dysregulation. The incidence of alternative splicing among neuropeptides and neuropeptide receptor genes, critical cell-cell signaling molecules, associated with behavior may compromise the replicability of reported maternal immune activation effects at the gene level. This study aims to advance the understanding of the effect of maternal immune activation on transcript isoforms of the neuropeptide system (including neuropeptide, receptor and connecting pathway genes) underlying behavior disorders later in life. Recognizing the wide range of bioactive peptides and functional receptors stemming from alternative splicing, we studied the effects of maternal immune activation at the transcript isoform level on the hippocampus and amygdala of three-week-old pigs exposed to maternal immune activation due to viral infection during gestation. In the hippocampus and amygdala, 29 and 9 transcript isoforms, respectively, had maternal immune activation effects (P-value < 0.01). We demonstrated that the study of the effect of maternal immune activation on neuropeptide systems at the isoform level is necessary to expose opposite effects among transcript isoforms from the same gene. Genes were maternal immune activation effects have also been associated with neurodevelopmental and behavior disorders. The characterization of maternal immune activation effects at the transcript isoform level advances the understanding of neurodevelopmental disorders and identifies precise therapeutic targets.

The pork industry faces financial losses associated with outbreaks in porcine reproductive and respiratory virus (PRRSV). One type of loss stems from the effect of PRRSV-elicited maternal immune activation during gestation on the response of pigs to a second immune challenge later in life. The objective of this study was to assess the effects of PRRSV infection during gestation, a second immune challenge, and sex on pig behavior. Camborough gilts were inoculated with PRRSV or saline (Controls) during the last third of gestation. The piglets from these gilts were weaned at day 21, and were injected with Poly(I:C) to elicit a second immune challenge, similarly to a viral infection, or with Saline on day 60. Post injection, behaviors were recorded in 5-minute intervals for one hour by a trained experimenter. Recorded behaviors included laying, standing, and panting, and totaled 624 observations per behavior on 51 pigs from 10 gilts. The logistic generalized mixed effect model used to describe behavior included the effects of gilt and pig treatments, sex, and interactions, and accounted for the random effect of gilt, and repeated measurements within pig. Panting showed a significant gilt challenge-by-sex-by-pig treatment interaction effect (P < 0.031). The Poly(I:C) challenge had a significant and positive effect on panting among male pigs from Control gilts (P < 0.001), while this effect was not observed in pigs from PRRSV-treated gilts. Among female pigs challenged with Poly(I:C), PRRSV treatment has a significant effect on lateral laying (P < 0.015) and standing (P < 0.042). Pigs from PRRSV-treated gilts were less active than from Control gilts. Our results highlight that the effect on behaviors of immune challenge later in life depend on the exposure of pigs to PRRSV during gestation. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

The synthetic polyinosinic-polycytidylic acid Poly(I:C) is commonly used to mimic viral infection in rodents. A Poly(I:C) injection activates inflammatory receptors that recognize viral patterns. This immune-stimulating agent can be used to understand the effects of viral infection on pork production. The objective of this study was to compare the effects of multiple Poly(I:C) doses on sickness behaviors across time. Female and male pigs from the University of Illinois Swine herd, averaging 24.5 kg, were injected with 1 mg/kg or 0.5 mg/kg of Poly(I:C), or comparable volume of Saline at approximately 60 days of age. Post injection, sickness behaviors and activity levels were measured in 15-minute intervals by a trained experimenter for three hours. Observed sickness behaviors included vomiting, diarrhea, or shivering. The activity levels were scored 1 through 8 with lower levels indicating pigs running, medium levels indicating walking, and higher levels indicating laying behaviors. The binary sickness behavior and discrete activity levels from 130 observations from 10 pigs evenly distributed across sexes were analyzed using generalized mixed effect models. The model included the effects of Poly(I:C) dose, hour, sex, and interactions, and a repeated structure within pig. The administration of Poly(I:C) had a significant effect on activity level within one-hour post-injection, and Poly(I:C)-treated pigs had lower activity levels than the Saline group. The Poly(I:C) effect had a lower significance in males (P < 0.001) than females (P < 0.004). The Poly(I:C) treatment also had a significant effect on the probability of sickness behavior (P < 0.005). The probability of sickness behavior was 0.03, 0.18, and 0.23 for pigs treated with Saline, 0.5, and 1.0 mg/kg of Poly(I:C), respectively. Our results suggest that Poly(I:C) could be an effective agent in studying sickness response to viral infections in pigs. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.

Maternal immune activation (MIA) during gestation can alter immune responses of offspring to many stressors including weaning. Interleukin (IL)-10 and IL-6 are anti- and pro-inflammatory cytokines, respectively, released during the immune response. This study assessed the effects of MIA on piglets’ serum cytokine concentrations in response to weaning. Camborough gilts were inseminated at approximately 235 days of age and were inoculated intranasally on gestational day 76 with porcine reproductive and respiratory syndrome virus (PRRSV) (n = 11) or saline (n = 7). On postnatal day (PD) 21 approximately half of the piglets remained with sows (suckled) while the rest were weaned. Blood was collected from piglets prior to weaning on PD21 and at PD22. Serum concentrations of IL-6 and IL-10 were measured by ELISA. Cytokine concentrations were tested for fixed effects of gilt treatment (PRRSV or Control), piglet sex (female or male), management (suckled or weaned), sampling day (PD21 or PD22), interactions, and Normality assumptions. IL-10 presented a significant interaction between sex, gilt treatment, and management at PD22 (P < 0.05). Weaned males from PRRSV-treated gilts had higher IL-10 concentrations versus suckled males whereas females from PRRSV-treated gilts had comparable levels of IL-10 between management groups. Overall, IL-10 was higher in piglets from PRRSV-treated gilts relative to Control gilts and matching sex and management groups. Higher IL-6 concentrations were found in weaned males from PRRSV-treated gilts versus suckled males at PD22. This pattern between management groups was reversed in females from PRRSV-treated gilts. Among piglets from Control gilts, males possessed higher IL-6 concentrations versus females across management groups. These results suggest MIA can alter inflammatory cytokine concentrations differently between males and females in response to weaning. This altered response may reduce offspring resiliency to subsequent immune or management stressors. This study is supported by USDA NIFA AFRI, 2018-67015-27413.

Porcine reproductive and respiratory syndrome virus (PRRSV) costs the U.S. pork industry millions of dollars yearly due to production losses and management costs. The objective of this study was to evaluate the effects of maternal immune activation (MIA) elicited by PRRSV infection during gestation on the behavioral response of the offspring to subsequent immune challenge. On gestational day 76, PRRSV negative Camborough gilts (n = 6) were either inoculated with PRRSV or saline intranasally. Nineteen female and male piglets from these PRRSV positive and Control gilts were weaned at day 21 and subsequently housed in groups. At day 60, piglets were randomized by sex to receive either saline or an intraperitoneal injection of Poly(I:C) to represent immune challenge. Poly(I:C) is a synthetic polyinosinic-polycytidylic acid that mimics double-stranded RNA that activates host defense similar to viral infections by stimulating the release of cytotoxic cytokines and inducing interferon-gamma production. Post injection, binary laying, eating, vomiting, panting, and overall sickness behaviors were collected by instantaneous scan sampling at 1.5-minute intervals for 60 minutes by a trained experimenter blinded to the experimental design. Fisher’s exact test and Cochran-Mantel-Haenszel statistics were used to test the effects of gilt treatment, piglet challenge, and sex within and across factor levels. Male piglets from PRRSV-treated gilts exhibited significantly (P < 0.0455) fewer sickness behaviors than piglets from control gilts between 31 and 45 minutes post-challenge. Significant associations between gilt treatment and both panting (P < 0.0162) and overall sickness behavior (P < 0.0245), while controlling for sex and piglet challenge, were observed. This study characterized the MIA and sex-dependent nature of the effect Poly(I:C) immune challenge on behavior. Our results indicate that MIA is correlated to increased behavioral response to secondary viral infection in male offspring. This study is supported by USDA NIFA AFRI, grant number 2018-67015-27413.