Method for quantifying the Pasteurella multocida antigen adsorbed on aluminum hydroxide adjuvant in swine atrophic rhinitis vaccine.

Swine atrophic rhinitis is a disease caused by Pasteurella multocida and Bordetella bronchiseptica that affects pigs. Inactivated vaccines containing the toxins produced by Pasteurella multocida and Bordetella bronchiseptica have been widely used for the prevention of swine atrophic rhinitis. The efficacy of a vaccine is correlated with the amount of antigen present; however, the protective toxin of P. multocida bound to aluminum hydroxide, which is used as an adjuvant, can hinder the monitoring of the antigen concentration in the vaccine. This study assessed the applicability of a dot immunoassay as an antigen quantification method using monoclonal antibodies. This quantification method was able to detect the antigen with high specificity and sensitivity even when the antigen was bound to the adjuvant, and its application to vaccine products revealed a correlation between the amount of antigen present in the vaccine and the neutralizing antibody titers induced in pigs. The antigen quantification method presented in this study is a simple and sensitive assay capable of quantifying the amount of antigen present in a vaccine that can be used as an alternative quality control measure.

Redox balance and immunity of piglets pre- and post-E. coli challenge after treatment with hemp or fish oil, and vitamin E.

This study investigated the influence of polyunsaturated fatty acid composition and vitamin E supplementation on oxidative status and immune responses in weanling piglets pre- and post-E. coli challenge. Suckling piglets (n = 24) were randomly selected from two litters for an oral supplementation (1 mL/day) with fish oil or hemp oil and vitamin E supplementation (60 mg natural vitamin E/mL oil) from day 10 to 28 of age. At day 29 and 30 of age, each piglet was orally inoculated with 6.7 × 108 and 3.96 × 108 CFU of F4 and F18 E. coli, respectively. Blood was sampled from all piglets on day 28 before E. coli challenge and on day 35 of age to investigate immunological and oxidative stress markers in plasma. One week after weaning and exposure to E. coli, a general reduction in the α-tocopherol concentration and activity of GPX1 was obtained. Vitamin E supplementation lowered the extent of lipid peroxidation and improved the antioxidative status and immune responses after E. coli challenge. Hemp oil had the greatest effect on antioxidant enzyme activity. Provision of hemp oil and vitamin E to suckling piglets may reduce the incidence of post-weaning diarrhea.

Deletion of a 7-amino-acid region in the porcine epidemic diarrhea virus envelope protein induces higher type I and III interferon responses and results in attenuation in vivo.

Porcine epidemic diarrhea virus (PEDV) leads to enormous economic losses for the pork industry. However, the commercial vaccines failed to fully protect against the epidemic strains. Previously, the rCH/SX/2016-SHNXP strain with the entire E protein and the rCH/SX/2015 strain with the deletion of 7-amino-acid (7-aa) at positions 23-29 in E protein were constructed and rescued. The pathogenicity assay indicated that rCH/SX/2015 is an attenuated strain, but rCH/SX/2016-SHNXP belongs to the virulent strains. Then, the recombination PEDV (rPEDV-EΔaa23-aa29)strain with a 7-aa deletion in the E protein was generated, using the highly virulent rCH/SX/2016-SHNXP strain (rPEDV-Ewt) as the backbone. Compared with the rPEDV-Ewt strain, the release and infectivity of the rPEDV-EΔaa23-aa29 strain were significantly reduced in vitro, but stronger interferon (IFN) responses were triggered both in vitro and in vivo. The pathogenicity assay showed that the parental strain resulted in severe diarrhea (100%) and death (100%) in all piglets. Compared with the parental strain group, rPEDV-EΔaa23-aa29 caused lower mortality (33%) and diminished fecal PEDV RNA shedding. At 21 days, all surviving pigs were challenged orally with rPEDV-Ewt. No pigs died in the two groups. Compared with the mock group, significantly delayed and milder diarrhea and reduced fecal PEDV RNA shedding were detected in the rPEDV-EΔaa23-aa29 group. In conclusion, the deletion of a 7-aa fragment in the E protein (EΔaa23-aa29) attenuated PEDV but retained its immunogenicity, which can offer new ideas for the design of live attenuated vaccines and provide new insights into the attenuated mechanism of PEDV. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets and remains a large challenge to the pork industry. Unfortunately, no safe and effective vaccines are available yet. The pathogenesis and molecular basis of the attenuation of PEDV remain unclear, which seriously hinders the development of PEDV vaccines. This study found that the rPEDV carrying EΔaa23-aa29 mutation in the E protein induced significantly higher IFN responses than the parental virus, partially attenuated, and remained immunogenic in piglets. For the first time, PEDV E was verified as an IFN antagonist in the infection context and identified as a virulence factor of PEDV. Our data also suggested that EΔaa23-aa29 mutation can be a good target for the development of live attenuated vaccines for PEDV and also provide new perspectives for the attenuated mechanism of PEDV.

Nonstructural Protein 1 of Variant PEDV Plays a Key Role in Escaping Replication Restriction by Complement C3.

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1ß (IL-1ß) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1ß compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein ß (C/EBP-ß) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-ß phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.

Porcine dsRNA-binding protein Staufen1 facilitate dsRNA-RIG-I/MDA5 binding to activate the antiviral innate immunity response.

Innate immune system composed of pathogen pattern recognition receptors (PRRs) is the first barrier to recognize and defend viral invasion. Previously，the double-stranded RNA binding protein staufen1 (STAU1) was identified as an important candidate in regulating RIG-I/MDA5 signaling axis, which is the major cytosolic PRRs for initiating immune response to antagonize RNA viruses. However, the mechanism of STAU1 on RNA virus infection is still unclear. In the present study, we demonstrated that STAU1 is a highly conservative dsRNA-binding protein in human and mammals. The porcine STAU1 (pSTAU1) could bind to the PEDV original dsRNA in cytoplasm. Furthermore, pSTAU1 is a binding partner that can positively increase the combination of MDA5 and dsRNA in cells, but slightly on RIG-I-dsRNA binding. Moreover, knockdown pSTAU1 led to inhibition of poly(I:C)-stimulated, VSV and RIG-I/MDA5-induced activation of porcine INF-ß promotor activation. Overexpression pSTAU1 could positively suppress the VSV proliferation in 3D4/21 cells. In sum, our data identify pSTAU1 as a key component of RIG-I/MDA5 binding viral dsRNA required for innate antiviral immunity in swine. The novel findings provide a new insight into host sensing the RNA-viruses infection.

Immunoglobin G Sero-Dynamics Aided Host Specific Linear Epitope Identification and Differentiation of Infected from Vaccinated Hosts.

Differentiation of infected from vaccinated hosts (DIVH) is a critical step in virus eradication programs. DIVH-compatible vaccines, however, take years to develop, and are therefore unavailable for fighting the sudden outbreaks that typically drive pandemics. Here, we establish a protocol for the swift and efficient development of DIVH assays, and show that this approach is compatible with any type of vaccines. Using porcine circovirus 2 (PCV2) as the experimental model, the first step is to use Immunoglobin G (IgG) sero-dynamics (IsD) curves to aid epitope discovery (IsDAED): PCV2 Cap peptides were categorized into three types: null interaction, nonspecific interaction (NSI), and specific interaction (SI). We subsequently compared IsDAED approach and traditional approach, and demonstrated identifying SI peptides and excluding NSI peptides supports efficient diagnostic kit development, specifically using a protein-peptide hybrid microarray (PPHM). IsDAED directed the design of a DIVH protocol for three types of PCV2 vaccines (while using a single PPHM). Finally, the DIVH protocol successfully differentiated infected pigs from vaccinated pigs at five farms. This IsDAED approach is almost certainly extendable to other viruses and host species. IMPORTANCE Sudden outbreaks of pandemics caused by virus, such as SARS-CoV-2, has been determined as a public health emergency of international concern. However, the development of a DIVH-compatible vaccine is time-consuming and full of uncertainty, which is unsuitable for an emergent situation like the ongoing COVID-19 pandemic. Along with the development and public health implementation of new vaccines to prevent human diseases, e.g., human papillomavirus vaccines for cervical cancer; enterovirus 71 vaccines for hand, foot, and mouth disease; and most recently SARS-CoV-2, there is an increasing demand for DIVH. Here, we use the IsDAED approach to confirm SI peptides and to exclude NSI peptides, finally to direct the design of a DIVH protocol. It is plausible that our IsDAED approach is applicable for other infectious disease.

Fc-Mediated E2-Dimer Subunit Vaccines of Atypical Porcine Pestivirus Induce Efficient Humoral and Cellular Immune Responses in Piglets.

Congenital tremor (CT) type A-II in piglets is caused by an emerging atypical porcine pestivirus (APPV), which is prevalent in swine herds and a serious threat to the pig production industry. This study aimed to construct APPV E2 subunit vaccines fused with Fc fragments and evaluate their immunogenicity in piglets. Here, APPV E2Fc and E2ΔFc fusion proteins expressed in Drosophila Schneider 2 (S2) cells were demonstrated to form stable dimers in SDS-PAGE and western blotting assays. Functional analysis revealed that aE2Fc and aE2ΔFc fusion proteins could bind to FcγRI on antigen-presenting cells (APCs), with the affinity of aE2Fc to FcγRI being higher than that of aE2ΔFc. Moreover, subunit vaccines based on aE2, aE2Fc, and aE2ΔFc fusion proteins were prepared, and their immunogenicity was evaluated in piglets. The results showed that the Fc fusion proteins emulsified with the ISA 201VG adjuvant elicited stronger humoral and cellular immune responses than the IMS 1313VG adjuvant. These findings suggest that APPV E2 subunit vaccines fused with Fc fragments may be a promising vaccine candidate against APPV.

Characterization and evaluation of a recombinant multiepitope peptide antigen MAG in the serological diagnosis of Toxoplasma gondii infection in pigs.

BACKGROUND: Toxoplasmosis caused by Toxoplasma gondii is a serious disease threatening human and animal health. People can be infected with T. gondii by ingesting raw pork contaminated with cysts or oocysts. Serological test is a sensitive and specific method usually used for large-scale diagnosis of T. gondii infection in humans and animals (such as pigs). Commercial pig Toxoplasma antibody ELISA diagnostic kits are expensive, which limits their use; moreover, the wide antigen composition used in these diagnostic kits is still unclear and difficult to standardize. The multiepitope peptide antigen is a novel diagnostic marker, and it has potential to be developed into more accurate and inexpensive diagnostic kits. METHODS: The synthetic multiepitope antigen (MAG) cDNA encoding a protein with epitopes from five T. gondii-dominant antigens (SAG1, GRA1, ROP2, GRA4, and MIC3) was designed, synthesized, and expressed in Escherichia coli BL21 (DE3) strain. The recombinant protein was detected through western blot with pig anti-T. gondii-positive and -negative serum, and then IgG enzyme-linked immunosorbent assay (ELISA) named MAG-ELISA was designed. The MAG-ELISA was evaluated in terms of specificity, sensitivity, and stability. The MAG-ELISA was also compared with a commercial PrioCHECK® Toxoplasma Ab porcine ELISA (PrioCHECK ELISA). Finally, the trend of pig anti-T. gondii IgG levels after artificial infection with RH tachyzoites was evaluated using MAG-ELISA and two other ELISA methods (rMIC3-ELISA and PrioCHECK ELISA). RESULTS: MAG antigen could be specifically recognized by pig anti-T. gondii-positive but not -negative serum. MAG-ELISA showed high diagnostic performance in terms of specificity (88.6%) and sensitivity (79.1%). MAG-ELISA could be used for detecting anti-T. gondii IgG in the early stage of T. gondii infection in pigs (at least 7 days after artificial infection). CONCLUSIONS: Our results suggest that MAG antigen can be applied to specifically recognize anti-T. gondii IgG in pig, and MAG-ELISA has the potential for large-scale screening tests of T. gondii infection in pig farms and intensive industries.

Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs.

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

Swine as biomedical animal model for T-cell research-Success and potential for transmittable and non-transmittable human diseases.

Swine is biologically one of the most relevant large animal models for biomedical research. With its use as food animal that can be exploited as a free cell and tissue source for research and its high susceptibility to human diseases, swine additionally represent an excellent option for both the 3R principle and One Health research. One of the previously most limiting factors of the pig model was its arguably limited immunological toolbox. Yet, in the last decade, this toolbox has vastly improved including the ability to study porcine T-cells. This review summarizes the swine model for biomedical research with focus on T cells. It first contrasts the swine model to the more commonly used mouse and non-human primate model before describing the current capabilities to characterize and extend our knowledge on porcine T cells. Thereafter, it not only reflects on previous biomedical T-cell research but also extends into areas in which more in-depth T-cell analyses could strongly benefit biomedical research. While the former should inform on the successes of biomedical T-cell research in swine, the latter shall inspire swine T-cell researchers to find collaborations with researchers working in other areas - such as nutrition, allergy, cancer, transplantation, infectious diseases, or vaccine development.

Recombinant pseudorabies virus with gI/gE deletion generated by overlapping polymerase chain reaction and homologous recombination technology induces protection against the PRV variant PRV-GD2013.

BACKGROUND: Since 2011, numerous highly virulent and antigenic variant viral strains have been reported in pigs that were vaccinated against the swine pseudorabies virus. These infections have led to substantial economic losses in the Chinese swine industry. RESULTS: This study, constructed a novel recombinant vaccine strain with gI/gE deletion (PRV-GD2013-ΔgI/gE) by overlapping PCR and homologous recombination technology. The growth curves and plaque morphology of the recombinant virus were similar to those of the parental strain. However, PRV-GD2013-ΔgI/gE infection was significantly attenuated in mice compared with that of PRV-GD2013. Two-week-old piglets had normal rectal temperatures and displayed no clinical symptoms after being inoculated with 105 TCID50 PRV-GD2013-ΔgI/gE, indicating that the recombinant virus was avirulent in piglets. Piglets were immunized with different doses of PRV-GD2013-ΔgI/gE, or a single dose of Bartha-K61 or DMEM, and infected with PRV-GD2013 at 14 days post-vaccination. Piglets given high doses of PRV-GD2013-ΔgI/gE showed no obvious clinical symptoms, and their antibody levels were higher than those of other groups, indicating that the piglets were completely protected from PRV-GD2013. CONCLUSIONS: The PRV-GD2013-ΔgI/gE vaccine strain could be effective for immunizing Chinese swine herds against the pseudorabies virus (PRV) strain.

Establishment of inflammatory model induced by Pseudorabies virus infection in mice.

BACKGROUND: Pseudorabies virus (PRV) infection leads to high mortality in swine. Despite extensive efforts, effective treatments against PRV infection are limited. Furthermore, the inflammatory response induced by PRV strain GXLB-2013 is unclear. OBJECTIVES: Our study aimed to investigate the inflammatory response induced by PRV strain GXLB-2013, establish an inflammation model to elucidate the pathogenesis of PRV infection further, and develop effective drugs against PRV infection. METHODS: Kunming mice were infected intramuscularly with medium, LPS, and different doses of PRV-GXLB-2013. Viral spread and histopathological damage to brain, spleen, and lung were determined at 7 days post-infection (dpi). Immune organ indices, levels of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines, as well as levels of activity of COX-2 and iNOS were determined at 4, 7, and 14 dpi. RESULTS: At 105-106 TCID50 PRV produced obviously neurological symptoms and 100% mortality in mice. Viral antigens were detectable in kidney, heart, lung, liver, spleen, and brain. In addition, inflammatory injuries were apparent in brain, spleen, and lung of PRV-infected mice. Moreover, PRV induced increases in immune organ indices, ROS and NO levels, activity of COX-2 and iNOS, and the content of key pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, interferon-γ and MCP-1. Among the tested doses, 10² TCID50 of PRV produced a significant inflammatory mediator increase. CONCLUSIONS: An inflammatory model induced by PRV infection was established in mice, and 10² TCID50 PRV was considered as the best concentration for the establishment of the model.

Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis

BACKGROUND: Lawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate. RESULTS: Batch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers. CONCLUSIONS: Considering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.

Primary characterization of the immune responses in Tibetan pigs infected with Chinese Tibet isolate of Trichinella spiralis.

BACKGROUND: Trichinellosis, caused by Trichinella spiralis, is a serious foodborne parasitic zoonosis. Tibetan pig is an infrequent, endemic plateau pig species, mainly distributed in Tibet Plateau, China. Because of the free-range system, Tibetan pigs are at risk of infection with Trichinella. The present study aimed to primarily profile the characteristics of T. spiralis infection in Tibetan pigs, including IgG levels, larvae burdens, and cytokines. RESULTS: The immune responses to Chinese Tibet T. spiralis isolate infection in Tibetan pigs with different doses were investigated in a tracking duration of 49 days. The muscle larvae per gram (lpg) were evaluated at 105 days post-infection (dpi). The results showed that the mean larval number of T. spiralis in Tibetan pigs increased with infective dose, with average lpg values of 3.5, 50.4 and 115.6 for Tibetan pigs infected with 200, 2,000, and 20,000 muscle larvae (ML) of T. spiralis. The anti-Trichinella IgG increased with inoculum dose and dpi, and peaked at 49 dpi. The kinetics of cytokines in the sera was detected by microarray, including interferon-γ (IFN-γ), interleukin (IL)-1ß, IL-8, IL-12, IL-4, IL-6, IL-10, Granulocyte-macrophage Colony Stimulating Factor (GM-CSF), tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-ß1. The Th1/Th2 mixed cytokines were detectable in all samples. Interleukin-12 demonstrated the highest concentration compared to other cytokines and peaked at 42 dpi. Almost all cytokines were maintained at a high level at 42 dpi. Additionally, we also report a Trichinella seropositive rate of 43.9 % (18 out of 41) from field samples of Tibetan pigs. CONCLUSIONS: The present study showed an increased Th1/Th2 mixed cytokines in Tibetan pigs elicited by T. spiralis. The high seroprevalence of Trichinella infection in field samples of Tibetan pigs further raises serious concern for the prevention and control of trichinellosis in this host for public health safety.

Occurrence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in pigs in the State of Pará, Brazil / Ocorrência de anticorpos contra Toxoplasma gondii e Neospora caninum em suínos no estado do Pará, Brasil

Abstract We investigated the occurrence of Toxoplasma gondii and Neospora caninum antibodies in pigs raised in the Northeast of Pará, Brazil. At Study I, convenience sampled 151 pigs at two slaughterhouses, with and without state inspection; and Study II, which assessed 159 pigs with probabilistic sampling from nine pig farms. Serological analysis was performed using indirect fluorescent antibody test for T. gondii and N. caninum with a cutoff of 64 and 50, respectively. Overall, 6.77% pigs were seropositive for T. gondii and 5.16% for N. caninum. In Study I, pigs slaughtered with and without state inspection presented similar occurrence for both coccidia (p>0.05). Study II found an association between N. caninum seropositivity and sludge discarded into the soil, feeding pigs with animal-based protein, subsistence system, and absence of nipple drinkers. No association was found for T. gondii. Pigs from Pará are a potential source of T. gondii infection to humans. To our best knowledge, this is the first study to report anti-N. caninum antibodies in the serum of pigs in Pará State, Brazilian Amazon.

Resumo Foi investigada a ocorrência de anticorpos contra Toxoplasma gondii e Neospora caninum em suínos criados no nordeste do Pará, Brasil. No Estudo I, foram amostrados 151 porcos em dois matadouros, com e sem inspeção estadual. O Estudo II avaliou 159 suínos com amostragem probabilística de nove granjas de suínos. Para sorologia, utilizou-se o teste de imunofluorescência indireta para T. gondii e N. caninum com ponto de corte de 1:64 e 1:50, respectivamente. No geral, 6,77% dos suínos foram soropositivos para T. gondii e 5,16% para N. caninum. No Estudo I, suínos abatidos em matadouros com e sem inspeção estadual apresentaram ocorrência semelhante para ambos os coccídios (p> 0,05). Os animais amostrados de Belém, Benevides, Marituba, Bujaru, Castanhal e Igarapé-Miri foram positivos para T. gondii, enquanto os soropositivos para N. caninum foram encontrados em Belém, Bujaru, Castanhal e Santo Antônio do Tauá. O Estudo II encontrou associação entre soropositividade de N. caninum e esterco descartado no solo, alimentação dos suínos com proteína de origem animal, criação de subsistência e ausência de bebedores tipo "nipple". Não foi encontrada associação para T. gondii. A carne suína apresenta potencial risco de transmissão de T. gondii para os habitantes da região. De acordo com nosso conhecimento, este é o primeiro relato de anticorpos anti-N. caninum em suínos no estado do Pará, Amazônia brasileira.

Chlamydia trachomatis L2c Infection in a Porcine Model Produced Urogenital Pathology and Failed to Induce Protective Immune Responses Against Re-Infection.

The current study was designed to evaluate the pathogenesis, pathology and immune response of female genital tract infection with Chlamydia trachomatis L2c, the most recently discovered lymphogranuloma venereum strain, using a porcine model of sexually transmitted infections. Pigs were mock infected, infected once or infected and re-infected intravaginally, and samples were obtained for chlamydial culture, gross and microscopic pathology, and humoral and cell-mediated immunity. Intravaginal inoculation of pigs with this bacterium resulted in an infection that was confined to the urogenital tract, where inflammation and pathology were caused that resembled what is seen in human infection. Re-infection resulted in more severe gross pathology than primary infection, and chlamydial colonization of the urogenital tract was similar for primary infected and re-infected pigs. This indicates that primary infection failed to induce protective immune responses against re-infection. Indeed, the proliferative responses of mononuclear cells from blood and lymphoid tissues to C. trachomatis strain L2c were never statistically different among groups, suggesting that C. trachomatis-specific lymphocytes were not generated following infection or re-infection. Nevertheless, anti-chlamydial antibodies were elicited in sera and vaginal secretions after primary infection and re-infection, clearly resulting in a secondary systemic and mucosal antibody response. While primary infection did not protect against reinfection, the porcine model is relevant for evaluating immune and pathogenic responses for emerging and known C. trachomatis strains to advance drug and/or vaccine development in humans.

Ulva prolifera Extract Alleviates Intestinal Oxidative Stress via Nrf2 Signaling in Weaned Piglets Challenged With Hydrogen Peroxide.

Background: Ulva prolifera extract contains a variety of functional active substances. Whether these substances had any beneficial effects on the small intestine of weaned piglets under oxidative stress remained unknown. Method: We explored the effects of U. prolifera extract on oxidative stress and related mechanisms in weaned piglets and intestinal porcine epithelial cells (IPEC-J2) challenged with hydrogen peroxide. Results: U. prolifera extract was found to mainly consist of polyphenols and unsaturated fatty acids. U. prolifera extract increased total antioxidant capacity and superoxide dismutase (SOD) activity, while it decreased malondialdehyde content, in the serum of weaned piglets challenged with hydrogen peroxide. Moreover, U. prolifera extract increased mRNA expression of SOD and catalase, as well as the intestinal expression of nuclear NF-E2-related factor 2 (Nrf2), both in vitro and in vivo. Furthermore, U. prolifera extract decreased reactive oxygen species and improved mitochondrial respiration in IPEC-J2 cells treated with hydrogen peroxide. However, AMPK inhibition did not affect nuclear Nrf2 expression and only partially affected the effects of U. prolifera extract on oxidative stress. Conclusion: We suggest that U. prolifera extract alleviates oxidative stress via Nrf2 signaling, but independent of AMPK pathway in weaned piglets challenged with hydrogen peroxide. These results shed new insight into the potential applications of U. prolifera extract as a therapeutic agent for the prevention and treatment of oxidative stress-induced intestinal diseases.

Reduced rotavirus vaccine efficacy in protein malnourished human-faecal-microbiota-transplanted gnotobiotic pig model is in part attributed to the gut microbiota.

The low efficacy of human rotavirus (HRV) vaccines in low- and middle-income countries (LMIC) remains a major challenge for global health. Protein-calorie malnutrition (kwashiorkor) affects the gut microbiota and compromises immune development, leading to environmental enteropathy, vaccine failures, and increased susceptibility to enteric diseases in young children. Relationship between diet and reduced vaccine efficacy in developing countries is not well established; therefore, we investigated the interconnections between the host-microbiota-nutrition-HRV vaccine using HRV-vaccinated, human infant faecal microbiota (HIFM)-transplanted neonatal gnotobiotic pigs fed with a protein deficient or sufficient diet. The microbiota from faecal, intestinal (duodenum, ileum, jejunum, and colon), and systemic tissue (liver, spleen, and mesenteric lymph node [MLN]) samples was analysed before and after HRV challenge using MiSeq 16S rRNA sequencing. Overall, microbiota from deficient fed HIFM pigs displayed, compared to the sufficient group, significantly higher Shannon index, especially in the faeces and lower intestines; higher level of Proteus and Enterococcus, and lower level of Bifidobacterium, Clostridium, and Streptococcus in the three types of samples collected (P<0.05); and higher unique operational taxonomic units (OTUs), especially in the systemic tissues. Further, the multivariate analysis between microbiota and immunologic data showed that 38 OTUs at the genus level correlated (r2≤0.5 or ≥-0.5; P<0.05) with at least one host immune response parameter (regulatory [Tregs and transforming growth factor-ß], effectors [interferon (IFN)-γ+ CD4+ and CD8+ T cells, IFN-γ and interleukin (IL)-12], and inflammatory [tumour necrosis factor-α, IL-17 and IL-22]) and with opposite trends between diet groups. Differences described above were increased after HRV challenge. We demonstrated that a protein deficient diet affects the composition of the gut microbiota and those changes may further correlate with immune responses induced by HRV and perturbed by the deficient diet. Thus, our findings suggest that the reduced efficacy of HRV vaccine observed in Gn pig model is in part attributed to the altered microbiota composition.

Plant-Derived Trimeric CO-26K-Equivalent Epitope Induced Neutralizing Antibodies Against Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) is a causative agent of a highly infectious disease with a high mortality rate, especially in newborn piglets in Asian countries resulting in serious economic loss. The development of a rapid, safe, effective and cost-efficient vaccine is crucial to protect pigs against PEDV infection. The COE antigen is regarded to be a major target for subunit vaccine development against PEDV infection. The naturally assembled COE protein forms a homotrimeric structure. In the present study, we successfully produced a trimeric COE protein as a native structure by fusion with the C-terminal isoleucine zipper trimerization (GCN4pII) motif in Nicotiana benthamiana, with a high expression level shown via semi-quantified Western blots. Trimeric COE protein was purified via immobilized metal affinity chromatography (IMAC), and its trimeric structure was successfully demonstrated by a cross-linking reaction, and a native PAGE gel. A crude extract containing the COE trimer was used for evaluating immunogenicity in mice. After 1 and 2 booster immunizations, the crude extract containing trimeric COE elicited elevated PEDV-specific humoral responses, as demonstrated by ELISA and Western blot analyses. Notably, a virus-neutralizing antibody assay indicated that the neutralization activities of sera of mice vaccinated with the crude extract containing COE-GCN4pII were similar to those of mice vaccinated with a commercial vaccine. These results suggest that crude extract containing trimeric COE is a promising plant-based subunit vaccine candidate for PEDV prevention.

Immunogenicity and protective efficacy induced by an mRNA vaccine encoding gD antigen against pseudorabies virus infection.

Messenger RNA-based vaccines represent new tools with prophylactic and therapeutic potential characterized by high flexibility of application for infectious diseases. Pseudorabies virus (PRV) is one of the major viruses affecting the pig industry. PRV has serious effects in piglets, sows, and growing-fattening pigs and can lead to huge economic losses. In this study, an envelope glycoprotein D (gD) gene-based specific mRNA vaccine was generated, and a mouse model was used to investigate the protective efficacy of the vaccine. The gD mRNA vaccine and the recombinant plasmid pVAX-gD were transfected into BHK21 cells, and the antigenicity of the expressed proteins was detected by Western blot analysis. Groups of mice were vaccinated with the gD mRNA vaccine, pVAX-gD, and PBS. T cell immune responses were measured by flow cytometry or ELISA and serum neutralization tests every two weeks. The challenge with the PRV-XJ strain was performed eight weeks after the primary immunization, and the response was monitored for 15 days. The levels of specific and neutralizing antibodies in the gD mRNA vaccine group were significantly increased in 8 weeks compared to those in the control group, and cytokine levels, including that of IFN-γ/IL-2, were considerably higher than those in the control animal. Additionally, the proportion of CD4+/CD8+ cells in peripheral lymphocytes was remarkably increased. Our data demonstrate that mRNA is a promising and effective tool for the development of vaccines. The PRV-gD-based mRNA vaccine can elicit an efficient neutralizing antibody response and induce effective protection in mice in defense against PRV infection.