Immunization Of Pigs Research Articles

Characterization of the Protective Cellular Immune Response in Pigs Immunized Intradermally with the Live Attenuated African Swine Fever Virus (ASFV) Lv17/WB/Rie1.

Candidate vaccines against African swine fever virus (ASFV) based on naturally attenuated or genetically modified viruses have the potential to generate protective immune responses, although there is no consensus on what defines a protective immune response against ASFV. Studies, especially in sensitive host species and focused on unravelling protective mechanisms, will contribute to the development of safer and more effective vaccines. The present study provides a detailed analysis of phenotypic and functional data on cellular responses induced by intradermal immunization and subsequent boosting of domestic pigs with the naturally attenuated field strain Lv17/WB/Rie1, as well as the mechanisms underlying protection against intramuscular challenge with the virulent genotype II Armenia/07 strain. The transient increase in IL-8 and IL-10 in serum observed after immunization might be correlated with survival. Protection was also associated with a robust ASFV-specific polyfunctional memory T-cell response, where CD4CD8 and CD8 T cells were identified as the main cellular sources of virus-specific IFNγ and TNFα. In parallel with the cytokine response, these T-cell subsets also showed specific cytotoxic activity as evidenced by the increased expression of the CD107a degranulation marker. Along with virus-specific multifunctional CD4CD8 and CD8 T-cell responses, the increased levels of antigen experienced in cytotoxic CD4 T cells observed after the challenge in immunized pigs might also contribute to controlling virulent infection by killing mechanisms targeting infected antigen-presenting cells. Future studies should elucidate whether the memory T-cell responses evidenced in the present study persist and provide long-term protection against further ASFV infections.

Therapeutic prime/pull vaccination of HSV-2-infected guinea pigs with the ribonucleotide reductase 2 (RR2) protein and CXCL11 chemokine boosts antiviral local tissue-resident and effector memory CD4+ and CD8+ T cells and protects against recurrent genital herpes.

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and its effect on the frequency and severity of recurrent genital herpes in the recurrent herpes guinea pig model. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-2 ribonucleotide reductase 2 (RR2) protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 chemokines to recruit CD4+ and CD8+ T cells into the infected DRG and VM (Pull). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident and effector memory CD4+ and CD8+ T cells in both DRG and VM tissues. This was associated with less virus in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4+ and CD8+ T cells in reducing virus shedding at the vaginal site of infection and the severity of recurrent genital herpes and propose the novel prime-pull vaccine strategy to protect against recurrent genital herpes.IMPORTANCEThe present study investigates the novel prime/pull therapeutic vaccine strategy to protect against recurrent genital herpes using the latently infected guinea pig model. In this study, we used the strategy that involves immunization of herpes simplex virus type 2-infected guinea pigs using a recombinantly expressed herpes tegument protein-ribonucleotide reductase 2 (RR2; prime), followed by intravaginal treatment with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines to recruit T cells into the infected dorsal root ganglia (DRG) and vaginal mucosa (VM) (pull). We show that the RR2/CXCL11 prime-pull therapeutic vaccine strategy elicited a significant reduction in virus shedding in the vaginal mucosa and decreased the severity and frequency of recurrent genital herpes. This protection was associated with increased frequencies of functional tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells infiltrating latently infected DRG tissues and the healed regions of the vaginal mucosa. These findings shed light on the role of tissue-resident and effector memory CD4+ and CD8+ T cells in DRG tissues and the VM in protection against recurrent genital herpes and propose the prime-pull therapeutic vaccine strategy in combating genital herpes.

Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, β-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Spatio-temporal epidemiology of Japanese encephalitis virus infection in pig populations of eastern Uttar Pradesh, India, 2013-2022.

Japanese encephalitis (JE) is endemic in India. Although pigs are considered important hosts and sentinels for JE outbreaks in people, limited information is available on JE virus (JEV) surveillance in pigs. We investigated the spatio-temporal distribution of JEV seroprevalence and its association with climate variables in 4451 samples from pigs in 10 districts of eastern Uttar Pradesh, India, over 10 years from 2013 to 2022. The mean seroprevalence of IgG (2013-2022) and IgM (2017-2022) was 14% (95% CI 12.8-15.2) and 10.98% (95% CI 9.8-12.2), respectively. Throughout the region, higher seroprevalence from 2013 to 2017 was observed and was highly variable with no predictable spatio-temporal pattern between districts. Seroprevalence of up to 60.8% in Sant Kabir Nagar in 2016 and 69.5% in Gorakhpur district in 2017 for IgG and IgM was observed, respectively. IgG seroprevalence did not increase with age. Monthly time-series decomposition of IgG and IgM seroprevalence demonstrated annual cyclicity (3-4 peaks) with seasonality (higher, broader peaks in the summer and monsoon periods). However, most variance was due to the overall trend and the random components of the time series. Autoregressive time-series modelling of pigs sampled from Gorakhpur was insufficiently predictive for forecasting; however, an inverse association between humidity (but not rainfall or temperature) was observed. Detection patterns confirm seasonal epidemic periods within year-round endemicity in pigs in eastern Uttar Pradesh. Lack of increasing age-associated seroprevalence indicates that JEV might not be immunizing in pigs which needs further investigation because models that inform public health interventions for JEV could be inaccurate if assuming long-term immunity in pigs. Although pigs are considered sentinels for human outbreaks, sufficient timeliness using sero-surveillance in pigs to inform public health interventions to prevent JEV in people will require more nuanced modelling than seroprevalence and broad climate variables alone.

The transcriptomic insight into the differential susceptibility of African Swine Fever in inbred pigs

African swine fever (ASF) is a global threat to animal health and food security. ASF is typically controlled by strict biosecurity, rapid diagnosis, and culling of affected herds. Much progress has been made in developing modified live virus vaccines against ASF. There is host variation in response to ASF infection in the field and under controlled conditions. To better understand the dynamics underlying this host differential morbidity, whole transcriptome profiling was carried out in twelve immunized and five sham immunized pigs. Seventeen MHC homozygous inbred Large white Babraham pigs were sampled at three time points before and after the challenge. The changes in the transcriptome profiles of infected animals were surveyed over time. In addition, the immunization effect on the host response was studied as well among the contrasts of all protection subgroups. The results showed two promising candidate genes to distinguish between recovered and non-recovered pigs after infection with a virulent African swine fever virus (ASFV) pre-infection: HTRA3 and GFPT2 (padj < 0.05). Variant calling on the transcriptome assemblies showed a two-base pair insertion into the ACOX3 gene closely located to HTRA3 that may regulate its expression as a putative genomic variant for ASF. Several significant DGEs, enriched gene ontology (GO) terms, and KEGG pathways at 1 day and 7 days post-infection, compared to the pre-infection, indicate a significant inflammation response immediately after ASF infection. The presence of the virus was confirmed by the mapping of RNA-Seq reads on two whole viral genome sequences. This was concordant with a higher virus load in the non-recovered animals 7 days post-infection. There was no transcriptome signature on the immunization at pre-infection and 1 day post-infection. More samples and data from additional clinical trials may support these findings.

A Gently Processed Skim Milk-Derived Whey Protein Concentrate for Infant Formula: Effects on Gut Development and Immunity in Preterm Pigs.

Processing of whey protein concentrate (WPC) for infant formulas may induce protein modifications with severe consequences for preterm newborn development. The study investigates how conventional WPC and a gently processed skim milk-derived WPC (SPC) affect gut and immune development after birth. Newborn, preterm pigs used as a model of preterm infants were fed formula containing WPC, SPC, extra heat-treated SPC (HT-SPC), or stored HT-SPC (HTS-SPC) for 5 days. SPC contained no protein aggregates and more native lactoferrin, and despite higher Maillard reaction product (MRP) formation, the clinical response and most gut and immune parameters are similar to WPC pigs. SPC feeding negatively impacts intestinal MRP accumulation, mucosa, and bacterial diversity. In contrast, circulating T-cells are decreased and oxidative stress- and inflammation-related genes are upregulated in WPC pigs. Protein aggregation and MRP formation increase in HTS-SPC, leading to reduced antibacterial activity, lactase/maltase ratio, circulating neutrophils, and cytotoxic T-cells besides increased gut MRP accumulation and expression of TNFAIP3. The gently processed SPC has more native protein, but higher MRP levels than WPC, resulting in similar tolerability but subclinical adverse gut effects in preterm pigs. Additional heat treatment and storage further induce MRP formation, gut inflammation, and intestinal mucosal damage.

Immunisation of pigs with recombinant HEV vaccines does not protect from infection with HEV genotype 3

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide. Up to now, no approved treatment nor a globally licensed vaccine is available. Several recombinant HEV vaccines have been developed to protect against HEV infection in humans, including the commercially available Hecolin vaccine, which are mainly based on HEV genotype 1. However, the efficacy of these vaccines against other HEV genotypes, especially genotype 3 is unknown. In this study, we evaluated the protective efficacy of Hecolin® and a novel genotype 3-based vaccine p239(gt3) against HEV-3 in a pig infection model. Pigs were divided into three groups: one group was vaccinated with Hecolin®, the second group was vaccinated with p239(gt3), and the control group received no vaccine. All pigs were subsequently challenged with HEV genotype 3 to assess the effectiveness of the vaccines. Although all immunised animals developed a high titer of neutralizing antibodies, the results showed that both vaccine applications could not provide complete protection against HEV (gt3) infection: Two out of four animals of the Hecolin® group displayed even virus shedding, and viral RNA could be detected in bile and/or liver of three out of four animals in both vaccination groups. Only one out of four animals in each group was fully protected. Neither Hecolin® nor the novel p239(gt3) vaccine provided sufficient protection against genotype 3 infection. While Hecolin® only partial protected pigs from HEV shedding, the novel p239(gt3) vaccine was at least able to prevent infected pigs from virus shedding. The results highlight the need for further development of HEV vaccines that exhibit broad protection against multiple HEV genotypes and the use of appropriate animal infection models.

Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability

Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer.

Polyclonal Peptide Antisera.

Polyclonal antibodies are relatively easy to produce and may supplement monoclonal antibodies for some applications or even have some advantages.The choice of species for production of (peptide) antisera is based on practical considerations, including availability of immunogen (vaccine) and animals. Two major factors govern the production of antisera: the nature of adaptive immune responses, which take place over days/weeks and ethical guidelines for animal welfare.Here, simple procedures for immunization of mice, rabbits, sheep, goats, pigs, horses, and chickens are presented.

ИММУНОФЕРМЕНТНАЯ ТЕСТ-СИСТЕМА ДЛЯ ОЦЕНКИ НАПРЯЖЕННОСТИ ПОСТВАКЦИНАЛЬНОГО ИММУНИТЕТА К ВИРУСУ КЛАССИЧЕСКОЙ ЧУМЫ СВИНЕЙ

Classical swine fever (CSF), being a particularly dangerous transboundary disease of pigs of all breeds and ages, remains one of the most important threats to the global pig industry. An urgent task is the development of new recombinant vaccines that meet the requirements of the DIVA strategy. No less important is the issue of equipping preventive means with accurate and rapid diagnostic test systems that allow assessing the intensity of post-vaccination immunity in target animals. The purpose of this study is to construct an enzyme-linked immunosorbent test system for indicating antibodies to the CSF virus based on a recombinant antigen. Specific components of the test system were obtained using genetic engineering and biochemical methods. Optimization of the ELISA protocol was carried out on a sample of immune and normal pig sera with variations in parameters (time and temperature of sensitization, working concentrations of specific components, reaction time). Determination of the analytical characteristics of the test system included tests for specificity, sensitivity limit, positive-negative threshold, and intra-laboratory reproducibility. It was found that the following parameters are optimal for performing ELISA: sensitizing antigen concentration – 50 ng per well, sensitization time – 3 hours at 37 °C or 12 hours at 4 °C; dilution of the antispecies conjugate is 1:50000, binding time to the enzymatic label is 45 min. The analytical sensitivity limit of the test system was 190 ng/ml for protein, the intra-laboratory reproducibility of results between wells of one plate was no less than 95 %, between individual plates was no less than 93 %. The developed ELISA test system, which has high sensitivity and specificity, can be recommended for assessing the effectiveness of vaccination against CSF.

An Alternative Serological Measure for Assessing Foot-and-Mouth Disease Vaccine Efficacy against Homologous and Heterologous Viral Challenges in Pigs.

To analyze the relationship between homologous and heterologous serological titers of immunized pigs and their protection statuses against FMD virus challenges, in the present study, the correlation between the virus neutralization titers at 21 and 28 dpv and the protection statuses at 28 dpv against challenge with FMD virus was analyzed using data sets comprising five different combinations of homologous or heterologous challenge experiments in pigs vaccinated with type O (n = 96), A (n = 69), and Asia 1 (n = 74). As a result, the experiments were divided into three groups (21D-1, 21D-2, and 21D-3) in the 21-dpv model and two groups (28D-1 and 28D-2) in the 28-dpv model. Each response curve of groups 21D-1 and 21D-2 in the 21-dpv model was very similar to each curve of groups 28D-1 and 28D-2 in the 28-dpv model, respectively, even though there was an exceptional extra group (21D-3) in the 21-dpv model. The average titers estimating 0.75 probability of protection ranged from 1.06 to 1.62 log10 in the 21-dpv model and from 1.26 to 1.64 log10 in the 28-dpv model. In summary, we demonstrated that the serological method is useful for predicting the homologous and heterologous protection statuses of vaccinated pigs.

Glycoprotein 5-Derived Peptides Induce a Protective T-Cell Response in Swine against the Porcine Reproductive and Respiratory Syndrome Virus.

We analyzed the T-cell responses induced by lineal epitopes of glycoprotein 5 (GP5) from PRRSV to explore the role of this protein in the immunological protection mediated by T-cells. The GP5 peptides were conjugated with a carrier protein for primary immunization and booster doses. Twenty-one-day-old pigs were allocated into four groups (seven pigs per group): control (PBS), vehicle (carrier), PTC1, and PTC2. Cytokine levels were measured at 2 days post-immunization (DPI) from serum samples. Cytotoxic T-lymphocytes (CTLs, CD8+) from peripheral blood were quantified via flow cytometry at 42 DPI. The cytotoxicity was evaluated by co-culturing primed lymphocytes with PRRSV derived from an infectious clone. The PTC2 peptide increased the serum concentrations of pro-inflammatory cytokines (i.e., TNF-α, IL-1β, IL-8) and cytokines that activate the adaptive cellular immunity associated with T-lymphocytes (i.e., IL-4, IL-6, IL-10, and IL-12). The concentration of CTLs (CD8+) was significantly higher in groups immunized with the peptides, which suggests a proliferative response in this cell population. Primed CTLs from immunized pigs showed cytolytic activity in PRRSV-infected cells in vitro. PTC1 and PTC2 peptides induced a protective T-cell-mediated response in pigs immunized against PRRSV, due to the presence of T epitopes in their sequences.

Evaluation of the humoral and mucosal immune response of a multiepitope vaccine against COVID-19 in pigs.

This study evaluated the immune response to a multiepitope recombinant chimeric protein (CHIVAX) containing B- and T-cell epitopes of the SARS-CoV-2 spike's receptor binding domain (RBD) in a translational porcine model for pre-clinical studies. We generated a multiepitope recombinant protein engineered to include six coding conserved epitopes from the RBD domain of the SARS-CoV-2 S protein. Pigs were divided into groups and immunized with different doses of the protein, with serum samples collected over time to determine antibody responses by indirect ELISA and antibody titration. Peptide recognition was also analyzed by Western blotting. A surrogate neutralization assay with recombinant ACE2 and RBDs was performed. Intranasal doses of the immunogen were also prepared and tested on Vietnamese minipigs. When the immunogen was administered subcutaneously, it induced specific IgG antibodies in pigs, and higher doses correlated with higher antibody levels. Antibodies from immunized pigs recognized individual peptides in the multiepitope vaccine and inhibited RBD-ACE2 binding for five variants of concern (VOC). Comparative antigen delivery methods showed that both, subcutaneous and combined subcutaneous/intranasal approaches, induced specific IgG and IgA antibodies, with the subcutaneous approach having superior neutralizing activity. CHIVAX elicited systemic immunity, evidenced by specific IgG antibodies in the serum, and local mucosal immunity, indicated by IgA antibodies in saliva, nasal, and bronchoalveolar lavage secretions. Importantly, these antibodies demonstrated neutralizing activity against SARS-CoV-2 in vitro. The elicited antibodies recognized individual epitopes on the chimeric protein and demonstrated the capacity to block RBD-ACE2 binding of the ancestral SARS-CoV-2 strain and four VOCs. The findings provide proof of concept for using multiepitope recombinant antigens and a combined immunization protocol to induce a neutralizing immune response against SARS-CoV-2 in the pig translational model for preclinical studies.

The Production of Recombinant African Swine Fever Virus Lv17/WB/Rie1 Strains and Their In Vitro and In Vivo Characterizations.

Lv17/WB/Rie1-Δ24 was produced via illegitimate recombination mediated by low-dilution serial passage in the Cos7 cell line and isolated on PAM cell culture. The virus contains a huge ~26.4 Kb deletion in the left end of its genome. Lv17/WB/Rie1-ΔCD-ΔGL was generated via homologous recombination, crossing two ASFV strains (Lv17/WB/Rie1-ΔCD and Lv17/WB/Rie1-ΔGL containing eGFP and mCherry markers) during PAM co-infection. The presence of unique parental markers in the Lv17/WB/Rie1-ΔCD-ΔGL genome indicates at least two recombination events during the crossing, suggesting that homologous recombination is a relatively frequent event in the ASFV genome during replication in PAM. Pigs infected with Lv17/WB/Rie1-Δ24 and Lv17/WB/Rie1/ΔCD-ΔGL strains have shown mild clinical signs despite that ASFV could not be detected in their sera until a challenge infection with the Armenia/07 ASFV strain. The two viruses were not able to induce protective immunity in pigs against a virulent Armenia/07 challenge.

Effect of the biological castration vaccine (Improvac®) on sex hormones and reproductive organs of pigs in Vietnam

The Improvac® vaccine has been shown to improve growth performance, meat quality, and sexual behavior in swine herds in several countries. This study initially investigated the ability of this vaccine to control sex hormones and the development of reproductive organs in Vietnamese crossbred pigs (Yorkshire – Landrace x Duroc). A total of 45 male piglets were randomly divided into three groups: vaccinated at 12 and 18 weeks old, surgically castrated males at 7 days old, and entire males. Meanwhile, 40 female piglets were randomly assigned to one of two groups: those vaccinated at 12 and 16 weeks of age or entire females. Serum from experimental pigs was collected monthly before and after vaccination until slaughter (25 weeks of age). At the time of slaughter, reproductive organs were obtained to assess changes at the macroscopic and microscopic features. Anti-GnRH antibodies in the immunized male and female pigs increased dramatically and remained for several weeks following a booster shot, while serum levels of sex hormones were significantly lower than in the entire group (P&lt;0.05). In addition, the reproductive organs of the immunized groups were negatively affected compared to the control groups. The male vaccinated group showed a significant reduction in the size of reproductive organs and irreversible disruption of spermatogenesis and testicular structure. The ovary and reproductive organs in the immunized female group revealed degradation in the size and ovarian follicle development. Taken together, the Improvac® vaccine can decrease sex hormones, thereby disrupting the development of reproductive organs in vaccinated male and female pigs.

Dietary supplementation with Acremonium terricola culture alters the gut microbial structure and improves the growth performance, antioxidant status, and immune function of weaning piglets

BackgroundAcremonium terricola is used in the feed of dairy animals to promote growth and control diseases. However, the effects of dietary supplementation with A. terricola on the gut microbial structure of weaning piglets remain poorly understood. Therefore, in this study, we investigated the effects of dietary supplementation with A. terricola culture (ATC) on the growth performance, antioxidant status, immunity, and gut environment of weaning piglets. Sixty piglets were fed a basal diet supplemented with 1 g ATC/kg of basal diet (experimental group). Another 60 piglets did not receive ATC (control group). The intervention lasted for 20 days.ResultsThe experimental group had higher daily weight gain and feed efficiency than did the control group. Significant increases were noted in the levels of serum insulin (P = 0.0018), insulin-like growth factor (P = 0.0018), triiodothyronine (P = 0.0031), immunoglobulin A (P < 0.0001), immunoglobulin M (P = 0.001), immunoglobulin G (P = 0.0001), and interferon γ (P < 0.0001) in the experimental group compared with the levels in the control group. Furthermore, ATC supplementation significantly reduced (P < 0.05) the relative abundance of Shuttleworthia, Succinivibrio, Roseburia, Ruminococcus, and Paludibacter but increased that of Phascolarctobacterium, Megasphaera, Faecalibacterium, and Prevotella in the experimental group compared with that in the control group. Notably, ATC supplementation significantly increased the relative abundance of Faecalibacterium prausnitzii (P < 0.05), which is involved in anti-inflammatory activities, gut barrier enhancement, and butyrate production.ConclusionsDietary supplementation with ATC may improve the growth performance, antioxidant status, immunity, and fecal microflora of weaning pigs.

Effect of Mixed Lactiplantibacillus plantarum- and Bacillus subtilis-Fermented Feed on Growth, Immunity, and Intestinal Health of Weaner Pigs

Bamei pigs, an eximious local breed reared on the Tibetan Plateau of China, are facing problems such as feed shortages, weaning stress, and antibiotic abuse. This study aimed to improve the quality of feed, growth performance, intestinal microbiota, and immunity of Bamei pigs through feeding with potentially probiotic-fermented feed. Different feeds were administered to weaned Bamei piglets for 60 days, creating the following five experimental treatment groups: basal feed group; Lactiplantibacillus plantarum-fermented-feed group; Bacillus subtilis-fermented-feed group; mixed-fermented-feed group; and antibiotic-supplemented-feed group. The results showed that the pH, neutral detergent fiber, and acid detergent fiber of the potentially probiotic-fermented feeds were significantly reduced; organic acids were produced; and Coliform bacteria, Clostridium, and aerobic bacteria were effectively inhibited. Feeding with potentially probiotic-fermented feed not only reduced the feed-conversion ratio but also improved immunity by increasing the production of anti-inflammatory cytokines, as well as decreasing pro-inflammatory cytokines and typical inflammatory pathways. The 16s rDNA high-throughput-sequencing results showed that probiotic-fermented feed improved the diversity of intestinal microbiota, inhibited the growth of the opportunistic pathogens Clostridium and Streptococcus, increased the relative abundance of Lactobacillus and Prevotella, and promoted gut health, demonstrating the promising application prospects of potentially probiotic-fermented feed.

PSXIII-8 Effects of Dietary Lactobacillus Reuteri on Immune Responses, Blood Profiles, Intestinal Morphology, and Ileal Gene Expression of Weaned Pigs

Abstract Weaning period is critical for gut health and immunity of pigs due to negative effect of weaning stress. Probiotics can be a potential alternative to antibiotics. However, there is limited information on potential mechanism of gut health and immunity of dietary Lactobacillus reuteri. This study was conducted to investigate the effects of dietary Lactobacillus reuteri on immune responses, blood profiles, intestinal morphology, and ileal gene expression of weaned pigs. A total of 48 weaned pigs [average initial body weight (BW) = 6.51 ± 0.87 kg; 21 days old] were allotted to 2 dietary treatments in a randomized complete block design (block: initial BW and sex). Dietary treatments had 6 replicate pens with 4 pigs per pen and fed for 4 weeks. Dietary treatments consisted of a basal weaner diet based on corn-soybean meal (CON) and CON supplemented with 0.02% Lactobacillus reuteri IDCC 3701 (LR). The LR was obtained from a commercial company (ILDONG Bioscience Co., Ltd., Pyeongtaek, Republic of Korea). Blood samples were collected from randomly selected one pig per pen on day 1, 7, 14, and 28. Serum was obtained from the blood sample to measure immune responses. On the last day of the experiment, the pigs were euthanized to collect small intestinal segments for intestinal morphology. Ileal mucosa were collected from ileal tissue for gene expression analysis. Measurements were serum cortisol, tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), interleukin-1β (IL-1β), and interleukin-6 (IL-6) using ELISA kits, blood profiles using an automated hematology analyzer, intestinal morphology using hematoxylin and eosin method, and ileal gene expression of tight junction proteins and inflammatory cytokines using qRT-PCR. Data were analyzed by using PROC GLM of SAS. Experimental unit was a pen. Statistical model for immune responses, blood profiles, and intestinal morphology included dietary treatments as a main effect and initial BW as a covariate. The t-test was used for the comparison of ileal gene expression. Pigs fed LR had drecreased (P &amp;lt; 0.05) serum TNF-α and IL-6 on day 7 and serum TGF-β1 on day 14 than those fed CON. In addition, pigs fed LR had greater (P &amp;lt; 0.05) hemoglobin concentration on day 7 than those fed CON, but had reduced (P &amp;lt; 0.05) hematocrit on day 14. Pigs fed LR had greater (P &amp;lt; 0.05) villus height to crypt depth ratio in duodenum than those fed CON. The LR upregulated (P &amp;lt; 0.05) claudin-1 gene compared with CON, but downregulated (P &amp;lt; 0.05) interleukin-8 and TNF-α genes. In conclusion, addition of dietary Lactobacillus reuteri in weaner diets improved intestinal morphology, modulated blood profiles and immune responses, and gene expression of inflammatory cytokines.

5 DPP Lecture: Investigating the Impact of Weaning and Biological Sex on Gut Development: Discovering New Targets for Gut Inflammation

Abstract Early life stress is known to have a profound impact on the development and function of the gut and immune system in various species, including pigs. In particular, the process of weaning is a significant source of stress in pigs and has been shown to negatively affect gut health and immunity. This presentation aims to examine the current knowledge on the impact of early life stress, particularly weaning, on gut development and immunity in pigs with a focus on chronic inflammation as a driver of disease risk. The presentation will also compare the immune system and gut development between female and male castrates, highlighting the influence of biological sex on gut health. Additionally, new data on potential targets for gut inflammation will be presented. The findings of this presentation will be analyzed in the context of pig production and the implications for enhancing gut health and immunity throughout production. Overall, this presentation will provide a comprehensive overview of the current understanding of the impact of early life stress on gut development and immunity in pigs and highlights key areas for future research.

Quadrivalent neuraminidase RNA particle vaccine protects pigs against homologous and heterologous strains of swine influenza virus infection

Influenza A virus in swine (IAV-S) continues to cause significant negative impact to both sows and growing pigs. The viral hemagglutinin (HA) and neuraminidase (NA) genes continue to evolve with HA diversifying at a faster rate than NA. Depending on country, whole inactivated virus (WIV) commercial and autogenous vaccines, as well as veterinary prescription vaccines targeting HA, are currently available. The use of these vaccines is focused on reducing virus and clinical signs in sows and to provide HA-specific maternally derived antibodies (MDA) to their suckling pigs. The deficiency in this strategy is that HA-MDA does not persist long enough to protect pigs through their growing phase from infection, and HA-MDA can interfere with effective pig immunization. This study evaluated the immunogenicity and efficacy of an adjuvanted, quadrivalent RNA Particle vaccine (Sequivity NA), currently licensed as Sequivity® IAV-S NA. This vaccine was formulated based on four NA antigens representing the major NA clades of IAV subtypes H1N1, H1N2 and H3N2 circulating in swine herds in the United States. In a series of trials, pigs were vaccinated twice, at three days and three weeks of age (WOA), followed by challenge with either homologous or heterologous IAV strains at 8 or 15 WOA. The Sequivity NA vaccine induced robust serum NA inhibition (NI) antibody and protected against IAV-S strains with homologous and heterologous NA to that of the vaccine. The magnitude and duration of nasal shedding was reduced in vaccinated-pigs challenged with either homologous or heterologous virus within the same NA clade. This NA-based RNA Particle vaccine avoids the known impact of HA-MDA on pig vaccination and provides a new tool to successfully reduce IAV-induced disease in the pig population.