Immunization Of Pigs Research Articles

Gilt development to improve offspring performance and survivability.

Methods for developing incoming replacement gilts can indirectly and directly influence survivability of their offspring. Indirectly, having proper gilt development reduces culling rates and mortality, which increases longevity and creates a more mature sow herd. Older sows are more likely to have greater immunity than gilts and therefore can pass this along to their pigs in both quantity and quality of colostrum and milk, thus improving piglet survivability. Directly, proper gilt development will maximize mammary gland development which increases colostrum and milk production leading to large, healthy pig. As for the developing gilt at birth, increasing colostrum intake, reducing nursing pressure, providing adequate space allowance, and good growth rate can increase the likelihood that gilts successfully enter and remain in the herd. Light birth weight gilts (<1 kg) or gilts from litters with low birth weight should be removed early in the selection process. Gilts should be weaned at 24 d of age or older and then can be grown in a variety of ways as long as lifetime growth rate is over 600 g/d. Current genetic lines with exceptional growth rate run the risk of being bred too heavy, reducing longevity. On the other hand, restricting feed intake at specific times could be detrimental to mammary development. In these situations, reducing diet amino acid concentration and allowing ad libitum feed is a possible strategy. Gilts should be bred between 135 and 160 kg and at second estrus or later while in a positive metabolic state to increase lifetime productivity and longevity in the herd. Once bred, gilts should be fed to maintain or build body reserves without becoming over-conditioned at farrowing. Proper body condition at farrowing impacts the percentage of pigs born alive as well as colostrum and milk production, and consequently, offspring performance and survivability. Combined with the benefit in pig immunity conferred by an older sow parity structure, gilt development has lasting impacts on offspring performance and survivability.

Adenovirus-Vectored African Swine Fever Virus pp220 Induces Robust Antibody, IFN-γ, and CTL Responses in Pigs.

African Swine Fever Virus (ASFV) poses a serious threat to the pork industry worldwide; however, there is no safe vaccine or treatment available. The development of an efficacious subunit vaccine will require the identification of protective antigens. The ASFV pp220 polyprotein is essential for virus structural integrity. This polyprotein is processed to generate p5, p34, p14, p37, and p150 individual proteins. Immunization of pigs with a cocktail of adenoviruses expressing the proteins induced significant IgG, IFN-γ-secreting cells, and cytotoxic T lymphocyte responses. Four predicted SLA-I binding nonamer peptides, namely p34161−169, p37859−867, p1501363−1371, and p1501463−1471, recalled strong IFN-γ+ PBMC and splenocyte responses. Notably, peptide p34161−169 was recognized by PBMCs isolated from 7/10 pigs and by splenocytes isolated from 8/10 pigs. Peptides p37859−867 and p1501363−1371 stimulated recall IFN-γ+ responses in PBMCs and splenocytes isolated from 8/10 pigs, whereas peptide p1501463−1471 recalled responses in PBMCs and splenocytes isolated from 7/10 to 9/10 pigs, respectively. The results demonstrate that the pp220 polyprotein contains multiple epitopes that induce robust immune responses in pigs. Importantly, these epitopes are 100% conserved among different ASFV genotypes and were predicted to bind multiple SLA-I alleles. The outcomes suggest that pp220 is a promising candidate for inclusion in a prototype subunit vaccine.

Plant derived Nano-11 particle adsorbed with stimulator of interferon genes adjuvant and split influenza virus antigens elicits the cross-protective immunity in pigs

Abstract We evaluated the performance of split swine influenza A virus (SwIAV) H1N2 antigens with a plant-derived nanoparticle adjuvant (Nano-11) or its combination with a synthetic STING agonist ADU-S100 (NanoS100). Specific pathogen free pigs were vaccinated twice via intramuscular (IM) and intradermal (ID) routes and challenged with a virulent heterologous H1N1. Animals vaccinated IM or ID with H1N2-ADU-S100-Nano-11 had significantly increased cross-reactive IgG and IgA titers in serum, nasal secretion and bronchoalveolar lavage fluid. There was a significant increase of IFNγ+ effector memory T-helper/memory cells and late effector cytotoxic T cells (CTLs), IL-17A+ central and effector memory T-helper/memory cells, IL-17A+ early effector CTLs, and CXCL10+ plasmacytoid dendritic cells in the PBMCs of the ID injected H1N2-ADU-S100-Nano-11 compared to H1N2-Nano-11 group. The frequency of IFNγ+ late effector CTLs and effector memory T-helper/memory cells and IL-17A+ late effectorCTLs, as well as in lungs CXCL10+ plasmacytoid dendritic cells was increased in PBMCs of IM injected H1N2-ADUS100-Nano-11 pigs. Increased expression of IL-4 and IL-6 mRNA was observed in tracheobronchial lymph nodes of IM Nano-11 based vaccinates following challenge. Furthermore, the challenge virus load in the lungs and nasal passage was undetectable in IM H1N2+ADU-S100-Nano-11 vaccinates by day 6 post infection along with reduced macroscopic lung lesions. In conclusion, despite vast genetic difference (77% HA gene identity) between the H1N2 and H1N1 SwIAV, the NanoS100 adjuvanted vaccine elicited cross protective immune responses, suggesting the potential role of this combination adjuvant in inducing cross-protective immunity in pigs. Supported by USDA National Institute of Food and Agriculture, AFRI project 2019-67015-29814

Comparative Study on Jejunal Immunity and Microbial Composition of Growing-Period Tibetan Pigs and Duroc × (Landrace × Yorkshire) Pigs.

The gut microbiota plays vital roles in metabolizing nutrient, maintaining the intestinal epithelial barrier but also in modulating immunity. Host genetics and the pig breed are implicated in shaping gut microbiota. Tibetan pig is a unique native Chinese breed and has evolved to manifest a strong disease resistance. However, the immunity and microbiota of growing Tibetan (TP) pigs were still rarely understood. The jejunal immunity phenotype and microbial composition of TP and Duroc × (Landrace × Yorkshire) (DLY) pigs were explored through immunohistochemistry and 16S rRNA sequencing. Higher scores of clusters of differentiation 4 (CD4+) and Toll-like receptor 9 (TLR9) were observed in TP pigs than those of DLY pigs (p < 0.05), as were Interleukin 10 (IL-10) and zonular occludens 1 (ZO-1) (p < 0.01). Similar levels of bacterial richness and diversity were found in the jejunal microbiota of the TP and DLY pigs. However, the TP pigs showed a significantly different microbiome compared to DLY pigs at the genus level (ANOSIM; p < 0.05). Pseudomonas, Stenotrophomonas, Phenylobacterium, and Sandaracinobacter were enriched in DLY pigs (p < 0.05), while the Lactobacillus and Solibacillus had higher abundances in TP pigs than DLY pigs (p < 0.05). Tibetan pigs have “healthier” intestinal microbial communities than DLY pigs. Close relationships were found between jejunal immune performance and the differential bacteria, Lactobacillus can enhance porcine jejunal immunity, while Stenotrophomonas will have a negative impact on porcine gut immunity.

Nonspecific protection of heat-inactivated Mycobacterium bovis against Salmonella Choleraesuis infection in pigs

Trained immunity is the capacity of innate immune cells to produce an improved response against a secondary infection after a previous unrelated infection. Salmonellosis represents a public health issue and affects the pig farming industry. In general, vaccination against salmonellosis is still facing problems regarding the control of distinct serovars. Therefore, we hypothesized that an immunostimulant based on heat inactivated Mycobacterium bovis (HIMB) could have an immune training effect in pigs challenged with Salmonella enterica serovar Choleraesuis (S. Choleraesuis) and decided to explore the amplitude of this non-specific immune response. For this purpose, twenty-four 10 days-old female piglets were randomly separated in three groups: immunized group (n = 10) received orally two doses of HIMB prior to the intratracheal S. Choleraesuis-challenge, positive control group (n = 9) that was only challenged with S. Choleraesuis, and negative control group (n = 5) that was neither immunized nor infected. All individuals were necropsied 21 days post-challenge. HIMB improved weight gain and reduced respiratory symptoms and pulmonary lesions caused by S. Choleraesuis in pigs. Pigs immunized with HIMB showed higher cytokine production, especially of serum TNFα and lung CCL28, an important mediator of mucosal trained immunity. Moreover, immunized pigs showed lower levels of the biomarker of lipid oxidation malondialdehyde and higher activity of the antioxidant enzyme superoxide dismutase than untreated challenged pigs. However, the excretion and tissue colonization of S. Choleraesuis remained unaffected. This proof-of-concept study suggests beneficial clinical, pathological, and heterologous immunological effects against bacterial pathogens within the concept of trained immunity, opening avenues for further research.

76 Awardee Talk: Maternal Immune Activation Impacts Offspring Immune Function but not Muscle

Abstract Gestational health challenges may influence growth performance and immunity of offspring pigs during postnatal life. In particular, porcine reproductive and respiratory syndrome virus (PRRSV) is endemic in the U.S. herd, but its effects on surviving piglets are largely unknown. Further, dietary supplementation with soy isoflavones lessened the severity of PRRSV infections in weaning and growing pigs. Therefore, the goals of this study were to determine the impact of maternal PRRSV infection on offspring muscle and immune development and the potential of isoflavones to mitigate those effects. Thirteen first parity gilts were randomly allotted to 1 of 3 treatments: fed a diet devoid of isoflavones and not infected (CON), fed the control diet and infected with PRRSV (POS), and fed the control diet supplemented with 1,500-ppm soy isoflavones and infected with PRRSV (ISF). Isoflavone supplementation reduced (P = 0.04) viral load in dams 21d after infection, but did not alter (P &amp;gt; 0.05) feed disappearance and rectal temperature. Piglet mortality was increased (P = 0.04) by PRRSV infection in dams, and surviving piglets were infected with PRRSV throughout the study. Interestingly, muscle and organ weights were not different (P &amp;gt; 0.05) among treatments at postnatal d (PND) 3 with the exception of relative liver weight which was increased (P = 0.04) in POS compared with CON, with ISF being intermediate. Infected litters were lighter (P = 0.02) at PND 21, likely due to postnatal infection. Interestingly, at PND 21 relative liver weight was increased (P &amp;lt; 0.01) in ISF compared with CON and POS. Muscle fiber number and size did not differ between treatments (P ≥ 0.39). Piglets born to infected dams had slower responses during innate immune stimulation (P &amp;lt; 0.01) and also had shifts in T cell populations compared with CON pigs (P ≤ 0.02), indicating an inadequate vaccine response. Overall, maternal infection altered offspring immune responses but not muscle fiber development. Isoflavone supplementation did not mitigate these effects.

109 Awardee Talk: Dietary Supplementation of Bacillus Amyloliquefaciens Influenced Systemic Immunity and Intestinal Microbiome of Weaned Pigs

Abstract Supplementation of various Bacillus spp. has shown to improve growth performance, immune responses, and gut health of newly weaned pigs. The present study investigated the effects of dietary supplementation of 109 CFU/kg of Bacillus (B.) amyloliquefaciens on growth performance, diarrhea, systemic immunity, and intestinal microbiome of weaned pigs with or without enterotoxigenic Escherichia coli (ETEC) challenge. An antibiotic treatment was also included in the experiment to compare the efficacy of B. amyloliquefaciens vs. carbadox (50 mg/kg). ETEC challenge reduced (P &amp;lt; 0.05) growth rate but increased (P &amp;lt; 0.05) the frequency of diarrhea in pigs throughout the experiment. Supplementation of B. amyloliquefaciens tended (P &amp;lt; 0.10) to increase growth performance but had limited impacts on diarrhea of weaned pigs. ETEC challenge increased (P &amp;lt; 0.05) white blood cell (WBC) count on d 7 and 21 post-inoculation (PI), while supplementation of B. amyloliquefaciens tended (P &amp;lt; 0.10) to reduce WBC on d 7 PI and reduced (P &amp;lt; 0.05) WBC on d 21 PI, compared with control. No differences were observed in performance, diarrhea, and systemic immunity of pigs when comparing B. amyloliquefaciens with carbadox. Supplementation of B. amyloliquefaciens and antibiotics had different impacts on intestinal microbiome. ETEC challenge increased (P &amp;lt; 0.05) relative abundance of Lactobacillus in feces on d 7 PI but reduced (P &amp;lt; 0.05) relative abundance of Prevotella in feces on d 21 PI. Prior ETEC challenge, Lachnospiraceae was less (P &amp;lt; 0.05) abundant in feces of pigs supplemented with B. amyloliquefaciens than pigs in carbadox group on d 0 before ETEC infection. The relative abundance of Enterobacteriaceae in fecal samples was the lowest (P &amp;lt; 0.05) in pigs supplemented with carbadox among all treatment on d 0. On d 14 and 21 PI, the relative abundance of Actinobacteria in feces was the lowest (P &amp;lt; 0.05) in carbadox treatment among all treatment of the experiment. Ileal digesta were also collected on d 21 PI to analyze intestinal microbiota diversity changes among treatments. The Shannon diversity index was greater (P &amp;lt; 0.05) in ileal digesta of pigs supplemented with B. amyloliquefaciens than pigs supplemented with carbadox. ETEC reduced (P &amp;lt; 0.05) the relative abundance of Firmicutes and Lactobacillus and increased (P &amp;lt; 0.05) the relative abundance of Proteobacteria and Lachnospiraceae in ileal digesta. Under ETEC challenge, pigs supplemented with B. amyloliquefaciens had lower (P &amp;lt; 0.05) abundance of Firmicutes and Clostridiaceae and greater (P &amp;lt; 0.05) abundance of Lachnospiraceae in the ileum than pigs supplemented with carbadox. In conclusion, supplementation of B. amyloliquefaciens had similar impacts on growth performance and systemic immunity but had different impacts on intestinal microbiota of weaned pigs in comparison to carbadox.

Vaccine with bacterium‐like particles displaying HIV‐1 gp120 trimer elicits specific mucosal responses and neutralizing antibodies in rhesus macaques

SummaryPreclinical studies have shown that the induction of secretory IgA (sIgA) in mucosa and neutralizing antibodies (NAbs) in sera is essential for designing vaccines that can effectively block the transmission of HIV‐1. We previously showed that a vaccine consisting of bacterium‐like particles (BLPs) displaying Protan‐gp120AE‐MTQ (PAM) could induce mucosal immune responses through intranasal (IN) immunization in mice and NAbs through intramuscular (IM) immunization in guinea pigs. Here, we evaluated the ability of this vaccine BLP‐PAM to elicit HIV‐1‐specific mucosal and systemic immune responses through IN and IM immunization combination strategies in rhesus macaques. First, the morphology, antigenicity and epitope accessibility of the vaccine were analysed by transmission electron microscopy, bio‐layer interferometry and ELISA. In BLP‐PAM‐immunized macaques, HIV‐1‐specific sIgA were rapidly induced through IN immunization in situ and distant mucosal sites, although the immune responses are relatively weak. Furthermore, the HIV‐1‐specific IgG and IgA antibody levels in mucosal secretions were enhanced and maintained, while production of serum NAbs against heterologous HIV‐1 tier 1 and 2 pseudoviruses was elicited after IM boost. Additionally, situ mucosal responses and systemic T cell immune responses were improved by rAd2‐gp120AE boost immunization via the IN and IM routes. These results suggested that BLP‐based delivery in combination with the IN and IM immunization approach represents a potential vaccine strategy against HIV‐1.

Evaluation of the prophylactic potential of non-enterotoxigenic Escherichia coli (non-ETEC) vaccine immunization and dietary mannan oligosaccharide competitive exclusion benefits against ETEC infections in weaned pigs

Enterotoxigenic Escherichia coli (ETEC) strains expressing F4 and F18 fimbriae are the most common causative agents of post-weaning diarrhoea (PWD) in pigs. The growing global restriction on the use of antibiotics in food animals has encouraged research into the development of nutritional and feeding strategies as well as vaccination against PWD. The aim of this study was to evaluate the efficacy of a live oral F4ac+ F18ac+ non-ETEC vaccine candidate (VAC) to stimulate gut and systemic cellular immunity in 4-week old pigs over 5 weeks following immunization. The onset and duration of protective immunity against on-farm occurring PWD, growth performance, diarrhoea scoring and mortality, as well as the phenotypic proportions of immune cells, were determined. Faecal and ileal samples were taken for determining the microbial composition or phenotyping of naïve/memory T cells. Also, the effect of prebiotic supplement mannan oligosaccharide (MOS) in the prevention of small intestinal colonization by ETEC, and its potential adjuvanticity in combination with the vaccine (VAC+MOS) were assessed. The pigs supplemented with MOS or that received VAC had significantly higher body weight (BW) (P&lt;0.05) on Day 14, whereas the VAC+MOS treated pigs had significantly lower BW on Day 35. Treatment with VAC+MOS resulted in considerably reduced clinical PWD, in particular the incidence and severity of diarrhoea and mortality. The total bacterial load in the ileum was much lower in the pigs from all 3 principal groups (MOS, VAC, and VAC+MOS) than in the control (CON) group (19 x 107, 17 x 107 and 12 x 107 vs. 23 x 108 CFU/mL, respectively) on Day 35. The pigs from the principal groups had significantly higher proportions of tested immune cells (P&lt;0.05) on Days 28 and 35. The localization and frequency of naive CD45RA+ and memory CD45RC+ T lymphocytes indicated their different distribution patterns within particular tissue structures, such as the villi, crypts, epithelium, lamina propria and areas (interfollicular follicular and Peyer’s patches) of ileal mucosa. This may indicate their different functions in intestinal immune responses to intraluminal microbes and their products, vaccinal immunogens and/or immunomodulators/adjuvants. To conclude, active mucosal immunity is needed to protect pigs against PWD. Hence, oral vaccination of pigs against both F4 and F18 ETEC, in combination with prebiotic supplementation represents a sustainable, practical and effective approach in PWD control.

Genetic Characteristics and Pathogenicity of a Novel Porcine Epidemic Diarrhea Virus with a Naturally Occurring Truncated ORF3 Gene.

Porcine epidemic diarrhea virus (PEDV) is the major pathogen that causes diarrhea and high mortality in newborn piglets, with devastating impact on the pig industry. To further understand the molecular epidemiology and genetic diversity of PEDV field strains, in this study the complete genomes of four PEDV variants (HN2021, CH-HNYY-2018, CH-SXWS-2018, and CH-HNKF-2016) obtained from immunized pig farms in central China between 2016 to 2021 were characterized and analyzed. Phylogenetic analysis of the genome and S gene showed that the four strains identified in the present study had evolved into the subgroup G2a, but were distant from the vaccine strain CV777. Additionally, it was noteworthy that a new PEDV strain (named HN2021) belonging to the G2a PEDV subgroup was successfully isolated in vitro and it was further confirmed by RT-PCR that this isolate had a large natural deletion at 207–373 nt of the ORF3 gene, which has never been reported before. Particularly, in terms of pathogenicity evaluation, colostrum deprivation piglets challenged with PEDV HN2021 showed severe diarrhea and high mortality, confirming that PEDV HN2021 was a virulent strain. Hence, PEDV strain HN2021 of subgroup G2a presents a promising vaccine candidate for the control of recurring porcine epidemic diarrhea (PED) in China. This study lays the foundation for better understanding of the genetic evolution and molecular pathogenesis of PEDV.

Preparation of bovine viral diarrhea disease virus 1 virus-like particles and evaluation of its immunogenicity in a guinea pig model

In order to obtain virus-like particles (VLPs) for prevention of bovine viral diarrhea virus 1 (BVDV-1), the C-Erns-E1-E2 region was cloned into a pFastBacDaul vector for generating the recombinant Bacmid-BVDV-1 in DH10Bac Escherichia coli. The recombinant baculovirus Baculo-BVDV-1 was produced by transfecting the Sf9 cells with Bacmid-BVDV-1. The expressed protein and the assembled VLPs were determined by immunofluorescence, Western blotting and electron microscopy. Guinea pigs were immunized with inactivated VLPs coupled with the Montanide ISA-201 adjuvant. The immunogenicity of VLPs was evaluated by monitoring the humoral immune response with neutralizing antibody titer determination, as well as by analyzing the cell-mediated immune response with lymphocyte proliferation assay. The protective efficacy of VLPs was evaluated by challenging with 106 TCID50 virulent BVDV-1 strain AV69. The results showed that the recombinant Baculo-BVDV-1 efficiently expressed BVDV structural protein and form VLPs in infected Sf9 cells. The immunization of guinea pigs with VLPs resulted in a high titer (1:144) of neutralizing antibody, indicating an activated cellular immunity. Significantly lower viral RNA in the blood of the post-challenged immunized guinea pigs was observed. The successful preparation of BVDV VLPs with insect cell expression system and the observation of the associated immunogenicity may facilitate further development of a VLPs-based vaccine against BVD.

Construction and Immunogenicity of a Recombinant Pseudorabies Virus Variant With TK/gI/gE/11k/28k Deletion

In China, the re-emerging pseudorabies virus (PRV) variant has caused large-scale outbreaks of pseudorabies in swine herds with classical PRV vaccine immunization since late 2011. Here, a recombinant PRV with TK/gI/gE/11k/28k deletion was constructed based on variant HN1201 strain isolated in 2012, by the bacterial artificial chromosome infectious clones. Compared with the parental virus, the recombinant PRV rHN1201TK−/gE−/gI−/11k−/28k− showed a similar virus grown curve and exhibited smaller plaques. The vaccination of rHN1201TK−/gE−/gI−/11k−/28k− could elicit an earlier and higher level of gB antibody, and the neutralizing antibodies elicited by rHN1201TK−/gE−/gI−/11k−/28k− were effective against both PRV classical and variant strains. Clinically, the body temperature of the pigs immunized with rHN1201TK−/gE−/gI−/11k−/28k− was significantly lower than that of the classical PRV vaccine immunized pigs, and the recombinant PRV could provide effective protection against the challenge with the PRV variant. These results imply that the rHN1201TK−/gE−/gI−/11k−/28k− could be a promising vaccine candidate for the prevention of the current epidemic of pseudorabies in China.

EFFECT OF MYCOTOXINS IN FEEDS ON INDICATORS OF CELLULAR IMMUNITY IN PIGS

The article presents the material on the study of the effect of the maximum permissible concentrations of mycotoxins in feeds on the indicators of the cellular link of immunity in pigs. In the experiment, a long-term intake of mycotoxins was modeled by the artificial addition of fusariotoxins (T-2 toxin) and ochratoxins at a dose of 0.2 and 0.12 mg/kg of animal body weight, respectively, and feeding pregnant and lactating sows, and later piglets, with contaminated feeds. Against the background of the presence of the maximum permissible concentrations of mycotoxins in feeds, the pigs at the age of 30, 60, 90 days and twelve weeks demonstrated a decrease in the number of immune blood cells of T-lymphocytes, including T-suppressors and T-helpers. At the age of 90 days, there was a decrease in the level of T-lymphocytes by 6 times, as well as their populations, T-suppressors – by 4.9 times and T-helpers – by 6.4 times, which can affect the state of general resistance of animals and lead to infectious and non-infectious diseases.

Nano/microparticle Formulations for Universal Influenza Vaccines

Influenza affects millions of people worldwide and can result in severe sickness and even death. The best method of prevention is vaccination; however, the seasonal influenza vaccine often suffers from low efficacy and requires yearly vaccination due to changes in strain and viral mutations. More conserved universal influenza antigens like M2 ectodomain (M2e) and the stalk region of hemagglutinin (HA stalk) have been used clinically but often suffer from low antigenicity. To increase antigenicity, universal antigens have been formulated using nano/microparticles as vaccine carriers against influenza. Utilizing polymers, liposomes, metal, and protein-based particles, indicators of immunity and protection in mouse, pig, ferrets, and chicken models of influenza have been shown. In this review, seasonal and universal influenza vaccine formulations comprised of these materials including their physiochemical properties, fabrication, characterization, and biologic responses in vivo are highlighted. The review is concluded with future perspectives for nano/microparticles as carrier systems and other considerations within the universal influenza vaccine delivery landscape. Graphical

ELISpot Assay for the Detection of ASFV-Specific Interferon-Gamma (IFN-γ)-Producing Cells.

The enzyme-linked immunospot (ELISpot) assay is a technique of unparalleled sensitivity to determine the frequency of antigen-specific immune cells secreting an immunomodulatory mediator upon recall antigen stimulation, making it a valuable tool in vaccine research. Typically done in multi-well microplate format, it also allows a high-throughput analysis of numerous immune cell samples, e.g., from different donor subjects, especially with the help of automated plate readers and specialized software that currently exist in most laboratories. IFN-γ is a hallmark cytokine secreted especially by T-cell subsets in recall response to pathogens, and consequently the IFN-γ ELISpot assay is one of the most widely used. The cellular arm of the immune response is known to be fundamental in protection against virulent ASFV, and therefore this assay is frequently employed in ASFV vaccine research to evaluate the results from immunization experiments.The technique involves the use of plates with wells that have a membrane for base with a strong binding capacity for amino acids that thus can be densely coated with an antibody for IFN-γ. Upon adding cells and specific antigen or other control stimuli, responding cells will release IFN-γ that is captured by the antibody in close proximity and revealed using a second antibody (sandwich method) through either chromogenic or fluorescent methods, leading to the detection of a "spot" on the membrane for each positive cell. Here we detail our protocol to detect the frequency of ASFV antigen-specific IFN-γ-producing cells in immunized pig lymphocytes and give an example of a typical result using the technique.

MORPHOLOGICAL CHARACTERISTICS OF GUINEA PIGS BRONCHUS ASSOCIATED LYMPHOID TISSUE IN THE DYNAMICS OF EXPERIMENTAL OVALBUMIN-INDUCED ALLERGIC INFLAMMATION

The actual problem of modern medicine is the reaction of Bronchus associated lymphoid tissue in airways chronic allergic diseases, because the full functioning of the respiratory system depends on the state of its local immune system.&#x0D; The aim – to study morphological changes in Bronchus associated lymphoid tissue of guinea pigs in the dynamics of experimental ovalbumin-induced allergic inflammation.&#x0D; Material and Methods. We have studied the lung of 48 guinea pigs, using histological and morphometric methods, under conditions of experimental ovalbumin-induced allergic inflammation. To assess the structural and functional rearrangement of pulmonary lymphoid nodules, their diameter and number were determined.&#x0D; Results. We have shown that sensitization and allergization with ovalbumin leads to a significant structural and functional rearrangement of Bronchus associated lymphoid tissue of guinea pigs in the form of the elevation in their number and diameter. We found more pronounced morphological changes in the late period of development of allergic inflammation in peribronchial lymphoid nodules, confirmed by the maximum magnification factor – 2.6 (p*/**&lt;0.001) of the diameter of peribronchial lymphoid nodules in the 4th experimental group compared to the control.&#x0D; Conclusions. Sensitization and inhalation with ovalbumin leads to structural reorganization of Bronchus associated lymphoid tissue of guinea pigs in the form of the elevation in their number and diameter, had the unidirectional staged character depending on localization of lymphoid nodules and the duration of the experiment and claimed proliferative changes in the cellular adaptive link of local lung immunity of guinea pigs in late period of allergic inflammatory process.

Tick immunity using mRNA, DNA and protein-based Salp14 delivery strategies

Guinea pigs exposed to multiple infestations with Ixodes scapularis ticks develop acquired resistance to ticks, which is also known as tick immunity. The I. scapularis salivary components that contribute to tick immunity are likely multifactorial. An anticoagulant that inhibits factor Xa, named Salp14, is present in tick saliva and is associated with partial tick immunity. A tick bite naturally releases tick saliva proteins into the vertebrate host for several days, which suggests that the mode of antigen delivery may influence the genesis of tick immunity. We therefore utilized Salp14 as a model antigen to examine tick immunity using mRNA lipid nanoparticles (LNPs), plasmid DNA, or recombinant protein platforms. salp14 containing mRNA-LNPs vaccination elicited erythema at the tick bite site after tick challenge that occurred earlier, and that was more pronounced, compared with DNA or protein immunizations. Humoral and cellular responses associated with tick immunity were directed towards a 25 amino acid region of Salp14 at the carboxy terminus of the protein, as determined by antibody responses and skin-testing assays. This study demonstrates that the model of antigen delivery, also known as the vaccine platform, can influence the genesis of tick immunity in guinea pigs. mRNA-LNPs may be useful in helping to elicit erythema at the tick bite site, one of the most important early hallmarks of acquired tick resistance. mRNA-LNPs containing tick genes is a useful platform for the development of vaccines that can potentially prevent selected tick-borne diseases.

A Co-Association Network Analysis Reveals Putative Regulators for Health-Related Traits in Pigs.

In recent years, the increase in awareness of antimicrobial resistance together with the societal demand of healthier meat products have driven attention to health-related traits in livestock production. Previous studies have reported medium to high heritabilities for these traits and described genomic regions associated with them. Despite its genetic component, health- and immunity-related traits are complex and its study by association analysis with genomic markers may be missing some information. To analyse multiple phenotypes and gene-by-gene interactions, systems biology approaches, such as the association weight matrix (AWM), allows combining genome wide association study results with network inference algorithms. The present study aimed to identify gene networks, key regulators and candidate genes associated to immunocompetence in pigs by integrating multiple health-related traits, enriched for innate immune phenotypes, using the AWM approach. The co-association network analysis unveiled a network comprised of 3,636 nodes (genes) and 451,407 edges (interactions), including a total of 246 regulators. From these, five genes (ARNT2, BRMS1L, MED12L, SUPT3H and TRIM25) were selected as key regulators as they were associated with the maximum number of genes with the minimum overlapping (1,827 genes in total). The five regulators were involved in pathways related to immunity such as lymphocyte differentiation and activation, platelet activation and degranulation, megakaryocyte differentiation, FcγR-mediated phagocytosis and response to nitric oxide, among others, but also in immunometabolism. Furthermore, we identified genes co-associated with the key regulators previously reported as candidate genes (e.g., ANGPT1, CD4, CD36, DOCK1, PDE4B, PRKCE, PTPRC and SH2B3) for immunity traits in humans and pigs, but also new candidate ones (e.g., ACSL3, CXADR, HBB, MMP12, PTPN6, WLS) that were not previously described. The co-association analysis revealed new regulators associated with health-related traits in pigs. This approach also identified gene-by-gene interactions and candidate genes involved in pathways related to cell fate and metabolic and immune functions. Our results shed new light in the regulatory mechanisms involved in pig immunity and reinforce the use of the pig as biomedical model.

Lactobacillus rhamnosus LB1 Alleviates Enterotoxigenic Escherichia coli-Induced Adverse Effects in Piglets by Improving Host Immune Response and Anti-Oxidation Stress and Restoring Intestinal Integrity.

Enterotoxigenic Escherichia coli (ETEC) is a common enteric pathogen that causes diarrhoea in humans and animals. Lactobacillus rhamnosus LB1 (formerly named Lactobacillus zeae LB1) has been shown to reduce ETEC infection to Caenorhabditis elegans and Salmonella burden in pigs. This study was to evaluate the effect of L. rhamnosus LB1 on the gut health of lactating piglets that were challenged with ETEC. Six-four piglets at 7 days of age were equally assigned into 8 groups (8 piglets per group): 1) control group (basal diet, phosphate buffer saline); 2) CT group (basal diet + 40 mg/kg colistin); 3) LL group (basal diet + 1 × 107 CFU/pig/day LB1); 4) HL group (basal diet + 1 × 108 CFU/pig/day LB1); 5) ETEC group: (basal diet + ETEC challenged); 6) CT + ETEC group (basal diet + CT + ETEC); 7) LL + ETEC group (basal diet + 1 × 107 CFU/pig/day LB1 + ETEC); 8) HL + ETEC group (basal diet + 1 × 108 CFU/pig/day LB1 + ETEC). The trial lasted ten days including 3 days of adaptation. Several significant interactions were found on blood parameters, intestinal morphology, gene, and protein expression. ETEC infection disrupted the cell structure and biochemical indicators of blood, undermined the integrity of the intestinal tract, and induced oxidative stress, diarrhoea, intestinal damage, and death of piglets. The supplementation of L. rhamnosus LB1 alleviated ETEC’s adverse effects by reducing pig diarrhoea, oxidative stress, and death, modulating cell structure and biochemical indicators of blood, improving the capacity of immunity and anti-oxidation stress of pigs, and restoring their intestinal integrity. At the molecular level, the beneficial effects of L. rhamnosus LB1 appeared to be mediated by regulating functional related proteins (including HSP70, Caspase-3, NLRP3, AQP3, and AQP4) and genes (including RPL4, IL-8, HP, HSP70, Mx1, Mx2, S100A12, Nrf2, GPX2 and ARG1). These results suggest that dietary supplementation of L. rhamnosus LB1 improved the intestinal functions and health of piglets.

Comparative Effects of Compound Enzyme and Antibiotics on Growth Performance, Nutrient Digestibility, Blood Biochemical Index, and Intestinal Health in Weaned Pigs.

This experiment aims to explore the effects of compound enzyme preparation substituting chlortetracycline on growth performance, serum immune markers, and antioxidant capacity and intestinal health in weaned piglets. A total of twenty-four 28-day-old “Duroc × Landrace × Yorkshire” weaned piglets with an average initial weight of 7.25 ± 0.25 kg were randomly divided into three groups according to their body weight, with eight replicates in each group and one pig in each replicate. The three dietary treatments were basal diet (CON), basal diet + 1,000 mg/kg compound enzyme preparation (cellulase 4,000 IU/g, α-amylase 1,000 IU/g, β-glucanase 150 IU/g, and neutral protease 3,000 IU/g, CE), and basal diet + 75 mg/kg chlortetracycline (CTC). The animal experiment lasted for 28 days and was divided into two stages: the early stage (0–14 days) and the late stage (15–28 days). The results showed that (1) compared with the CON, the CE and CTC significantly increased the ADG of weaned piglets during the early and whole period of experiment (p < 0.05), decreased the F:G in the whole experiment period (p < 0.05), and diarrhea rate in the early stage (p < 0.01). (2) Compared with the CON, the apparent total tract digestibility of ADF and NDF was significantly increased in pigs fed the CE diet in the early and late stages of experiment (p < 0.05) with no significant difference compared with the CTC. (3) Compared with the CON, the concentrations of serum IgA and SOD in weaned piglets were significantly increased in the CE group in the early stage of the experiment (p < 0.05). (4) Compared with the CON group, the acetic acid, propionic acid, and total VFA contents in cecum and colon segments were elevated in the CE group (p < 0.05) with no significant difference compared with the CTC. (5) Compared with the CON group, the villus height of duodenum and jejunum and the ratio of villus height to recess depth of ileum were increased in the CE and CTC group (p < 0.05). (6) Compared with the CON group, the abundance of Lactobacillus significantly increased (p < 0.01) while the abundance of Escherichia coli decreased in the CE group and CTC group (p < 0.01). In conclusion, CE preparation instead of CTC can significantly improve the nutrient digestibility, the immunity, antioxidant capacity, and intestinal health of pigs, which may contribute to the improved growth performance of piglets.