Infected Pigs Research Articles

An Efficient, Organic Solvent-Free Method for Extraction and Concentration of Hepatitis E Virus from Pig Liver

Abstract The presence of the hepatitis E virus (HEV) in pork products, particularly in pig liver has been frequently described. However, a standardized method is not still available for the detection of HEV in foods, particularly in those difficult food matrixes such as pig meat and pork products. The aim of this study was to design, optimize and evaluate a new method of food-separation and virus concentration for HEV in pig liver samples. This method is based on organic flocculation and avoids the use of organic solvents. The virus recovery rates and analytical sensitivity of the method using murine norovirus MNV-1 as a surrogate were 73.6–82.2% and at least 1 × 103 TCID50 per g of liver in 100% of the replicates, respectively. Furthermore, this new methodology was validated by testing the presence of HEV RNA in naturally infected pig liver samples, comparing it with two other commonly used concentration methods. The new extraction method run satisfactory in comparison with the two reference methods; statistically equivalent (p < 0.05) to one of the methods used while presented statistically significant better results (p < 0.05) compared to the second method. Consequently, our results indicate that the new extraction method can be an adequate cost-effective and ecologically friendly alternative for the food separation and concentration of HEV RNA in pig liver samples.

Modified multiplex PCR for serotyping and pathotyping of Streptococcus suis.

Introduction. Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans who have been in close contact with infected pigs or contaminated pork-derived products. There is currently no consensus on the universal virulence factors or markers that can differentiate pathogenic from non-pathogenic or commensal S. suis isolates.Gap statement. A diagnostic tool for serotyping and pathotyping of S. suis is required for active public health surveillance and the One-Health approach.Aim. To improve the former multiplex PCR to serotyping all 29 recognized 'true' serotypes and distinguish pathogenic pathotypes using primers targeting the capsule and ROK pathogenic marker genes.Methodology. Four sets of multiplex PCRs were modified and improved to detect all 29 recognized serotypes of S. suis and distinguish their pathogenic pathotypes using the ROK gene.Results. This multiplex PCR allowed for the simultaneous amplification of S. suis-specific, serotype-specific and pathogenic pathotypes from the DNA of each serotype in each reaction. The accuracy, sensitivity, specificity, positive predictive value and negative predictive value of the pathogenic ROK marker genes were 84.7% (625/738), 96.4% (423/439), 67.6% (202/299), 81.4% (423/520) and 92.7% (202/218), respectively. There was a significant (P-value <0.001), high positive likelihood ratio [2.9 with 2.5-3.5 of 95% confidence interval (CI)] and a significant odds ratio (55.1 with 31.6-95.9 of 95 % CI), which indicated that the ROK gene could be used as the pathogenic pathotype marker. No cross-reactions were observed with other bacterial species.Conclusion. This modified multiplex PCR was able to distinguish 29 well-known serotypes and predicted the pathogenic pathotypes of S. suis isolates from humans and pigs in a single assay. It is useful for One-Health surveillance of human and pig isolates of S. suis.

Antiviral mechanism of Fuzhengjiedu San against porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a viral infectious disease that can cause infection in pigs of different ages. The condition known as porcine reproductive and respiratory syndrome poses a serious risk to the world's pig business and results in significant financial losses. Fuzhengjiedu San (FZJDS) is a traditional Chinese medicine compound, the main components include:Radix Isatidis, Radix Astragali and Herba Epimedii. It has been widely used in clinical and experimental studies, showing a wide range of biological activity. However, it is not clear whether FZJDS has anti-PRRSV activity. We observed that FZJDS had significant antiviral activity in Marc-145cells. And FZJDS could inhibit viral infection in the stages of viral internalization and replication. Furthermore, FZJDS can inhibit PRRSV replication by inhibiting the p53 signaling pathway to affect autophagy, and FZJDS can also inhibit PRRSV replication by inhibiting the PI3K/Akt pathway.We showed in this work that FZJDS inhibits PRRSV replication in vitro and offers a novel therapeutic approach for PRRSV infection.

Immunization and challenge trials in a murine model using different inactivated recombinant vaccines against H1N1 swine influenza virus circulating in Brazil.

In Brazil, at least four lineages of influenza A virus circulate pig population: 2009 H1N1 flu pandemic (pH1N1), human-seasonal origin H3N2, H1N1 and H1N2 (huH1 lineages) viruses. Studies related to the occurrence of swine influenza A virus (SIAV) in Brazilian herds have been detecting an increase of occurrence of huH1 lineages. This study aimed to construct recombinant vaccines against the huH1N1 virus and test the immunogens in a murine model. The virus was constructed by reverse genetics using plasmids encoding the HA and NA sequences from a wild huH1N1 virus isolated from an infected pig. Amplified virus was inactivated, and oil-in-water (OW) and gel polymer (GP) adjuvants were used to formulate the vaccines. C57Bl6 mice received two doses with 3weeks interval by the intramuscular route. Animals were randomly divided into 8 groups (G1-G8): G1 received OW vaccine and G2 PBS plus OW adjuvant; G3 received GP vaccine and G4 PBS plus GP adjuvant; G5 received the live virus by the intranasal route while G6 only PBS; G7 and G8 did not receive any treatment. Serum samples were collected before vaccination and after the first and second dose. Except for G8, three weeks post boost animals were challenged with a wild huH1N1 virus and observed for weight changes. After infection, bronchoalveolar lavage fluid (BALF) and lungs were collected from animals of each group for viral titers and immunohistochemistry (IHC) analysis, respectively. After booster, vaccinated groups seroconverted and the vaccines induced protection upon challenge. Reverse Genetics technique can be used to produce new and quickly updated swine influenza vaccines which is promising to control the virus in Brazilian herds. Future studies may focus on using the technology to produce multivalent recombinant vaccines against distinct strains of SIAVs circulating in Brazilian pig herds.

ELISA-assay for Pig Taenia solium Cysticercosis by using T18 recombinant antigen

Cysticercosis is caused by infection with the larval stage of tapeworm parasite Taenia solium. This zoonose is a major public health issue with high prevalence in Madagascar, around 21% and 7% to 20% respectively in swine and human. ELISA and Enzyme-linked Immuno-electro Transfer Blot (EITB) remained the main laboratory serological diagnostic tools available in the island. Four native glycoproteins are recognized by antibodies in a majority of sera from patients with cysticercosis and a similar pattern seems to be present in infected pigs. For a better management of this disease in Madagascar, the aim of this study is to set-up and validate the one majority glycoprotein antigen T18 (18KDa) for diagnosis test of pig cysticercosis, produced as recombinant protein. Using a cDNA library prepared from T. solium cysticerci obtained from different regions of Madagascar, we first analyzed the polymorphism of the T18 gene. Then, gene was cloned into the pMAL-c2X plasmid for expression as an N-terminal maltose-binding protein (MBP) fusion protein and C-terminal hexa-His tagged protein. Product protein was then validated by ELISA test of pig sera. Preliminary results show a strong conservation of T18 gene. And indeed larger amounts of soluble recombinant protein were obtained from these antigen. T18 recombinant-ELISA present 70% of sensitivity and 100% of specificity.

Study of the Immunologic Value of Trichinella spiralis recombinant Protein Tsp-LE

Background and Aim. Trichinellosis is a serious zoonotic disease, with frequent outbreaks reported across various regions in Europe and Asia. However, current diagnostic tests based on excretory-secretory antigens (ES-Ag) and somatic antigens (S-Ag) lack adequate specificity and sensitivity. Moreover, the production of these antigens is both labor-intensive and costly.Materials and Methods. The immunodiagnostic potential of a recombinant 11 kDa serine protease protein, previously characterized in earlier studies was assessed. This study detected infection in Balb/c mice as early as 14 days post infection with T. spiralis larvae, yielding no false-positive results and an average infection level of 61±5.3 larvae per mouse.Results. Comparative indirect ELISA analysis on mouse sera after the 3rd and 5th immunizations, alongside tests on S Ag and S-Ag using sera from infected rabbits and pigs, demonstrated the high diagnostic accuracy of the recombinant protein. The protein revealed strong specificity for trichinellosis detection, showing no cross reactivity with echinococcosis.Conclusion. These findings support using rTsp-LE as a reliable component in developing ELISA test systems for trichinellosis detection.

Transcriptional analysis reveals the suppression of RAD51 and disruption of the homologous recombination pathway during PEDV infection in IPEC-J2 cells

PEDV is a highly contagious enteric pathogen that can cause severe diarrhea and death in neonatal pigs. Despite extensive research, the molecular mechanisms of host’s response to PEDV infection remain unclear. In this study, differentially expressed genes (DEGs), time-specific coexpression modules, and key regulatory genes associated with PEDV infection were identified. The analysis revealed 2,275, 1,492, and 3,409 DEGs in infected vs. mock-treated pigs at 12 h, 24 h, and 48 h, respectively. Time series analysis revealed that the upregulated genes were involved mainly in antiviral pathways such as the viral defense response and the regulation of immune system processes. Protein–protein interaction network analysis identified the top 20 core genes in the interaction network, which included six upregulated genes (TFRC, SUOX, RMI1, CD74, IFIH1, and CD86) and 14 downregulated genes (FOS, CDC6, CDCA3, PIK3R2, TUFM, VARS, ASF1B, POLD1, MCM8, POLA1, CDC45, BCS1L, RAD51, and RPA2). In addition, GSEA enrichment analysis revealed that pathways such as DNA replication and homologous recombination involving RAD51, CDC6, and RPA2 were significantly inhibited during viral infection. Our findings not only reveal dynamic changes in the transcriptome profile of PEDV-infected IPEC-J2 cells but also provide novel insights into the mechanism of PEDV infection of the host.

A triple protein-based ELISA for differential detection of ASFV antibodies.

African swine fever (ASF) caused by the ASF virus (ASFV) is a severe and highly contagious viral disease that poses a significant threat to the global pig industry. As no vaccines or effective drugs are available to aid prevention and control, early detection is crucial. The emergence of the low-virulence ASFV strain not expressing CD2v/MGFs (ASFVΔCD2v/ΔMGFs) has been identified domestically and internationally and has even become an epidemic in China, resulting in a complex epidemic. The commercialized ASFV ELISA kits available can detect the presence of ASFV infection in pigs, but they are unable to distinguish wild-type ASFV from gene-deleted strains. The current published ELISA assays can distinguish between the wild-type and CD2v gene-deleted ASFV but cannot differentiate wild-type and MGF505 gene-deleted ASFV or CD2v and MGF505 double-gene deleted ASFV infection, posing new challenges for an effective prevention and control of ASFV. In this study, the ASFV-p30, ASFV-CD2v, and ASFV-MGF505 proteins were expressed using a prokaryotic expression system, and a triple protein-based ELISA antibody detection method based on these proteins was successfully established to effectively differentiate between wild-type ASFV and ASFVΔCD2v and/or ASFVΔMGF505 virus infection. This triple protein-based ELISA showed good analytical specificity without cross-reactivity with antibodies against PRRSV, CSFV, PRV, and PCV2. Moreover, it demonstrates remarkable analytical sensitivity by allowing the identification of clinical samples even at dilutions as high as 1:800. The coefficient of variation the intra-assay and inter-assay were below 5%, indicating strong repeatability and reproducibility. To evaluate the performance of the triple protein-based ELISA, a total of 59 clinical serum samples were detected using the triple protein-based ELISA. The results showed that 22 samples were positive for ASFV, of which 19 were ASFV wild-type, one was ASFVΔCD2v, and two were ASFVΔMGF505. Compared with the commercialized triplex qPCR kit, the triple protein-based ELISA exhibited high diagnostic sensitivity and diagnostic specificity. The test accuracy with the commercialized triplex qPCR kit was 98.31% (58/59), and the test accuracy with the commercialized ELISA kit was 96.61% (57/59). These results indicated that the developed triple protein-based ELISA performs well in detection and differentiation. Therefore, it will be useful for the ASFV serological differential diagnosis and epidemiology study.

A Quadruplex RT-qPCR for the Detection of African Swine Fever Virus, Classical Swine Fever Virus, Porcine Reproductive and Respiratory Syndrome Virus, and Porcine Pseudorabies Virus.

African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine pseudorabies virus (PRV) induce similar clinical signs in infected pigs, including hyperthermia, anorexia, hemorrhage, respiratory distress, neurological symptoms, and/or abortions in pregnant sows. The differential diagnosis of these diseases relies on laboratory examinations. In this study, a quadruplex RT-qPCR was established using four pairs of specific primers and probes aimed at the B646L (p72) gene of ASFV, the 5' untranslated region (5'UTR) of CSFV, the ORF6 gene of PRRSV, and the gB gene of PRV for the detection and differentiation of ASFV, CSFV, PRRSV, and PRV. The assay exhibited great sensitivity with limits of detection (LODs) of 134.585, 139.831, 147.076, and 142.331 copies/reaction for ASFV, CSFV, PRRSV, and PRV, respectively. The assay exclusively identified ASFV, CSFV, PRRSV, and PRV, yielding negative results for the other control swine viruses used in this study. The intra-assay and inter-assay coefficients of variation (CVs) were not higher than 1.12%, indicating good reproducibility of the assay. The quadruplex RT-qPCR assay was used to analyze 3116 clinical tissue samples from pigs in Guangxi province, China, from April 2023 to September 2024. ASFV, CSFV, PRRSV, and PRV had positivity rates of 10.84% (338/3116), 0.80% (25/3116), 14.92% (465/3116), and 1.38% (43/3116), respectively, demonstrating a coincidence rate of ≥99.45% with the previously described RT-qPCR assays, which were also used to test these same samples. The established assay was rapid, sensitive, and accurate in detecting and differentiating ASFV, CSFV, PRRSV, and PRV.

Cloning genes of p49, p72, pe199l, pe248r and cd2vinto &lt;i&gt;Pichia Pastoris&lt;/i&gt; gs115

African swine fever (ASF), caused by the African swine fever virus (ASFV), poses a significant threat to pig populations worldwide. ASFV is a double-stranded DNA virus. In recent years, 54 structural proteins and more than 100 proteins have been involved in viral infection found in macrophages of diseased pigs. Among these, p49 and p72 are essential capsid proteins crucial for forming the viral capsid. Additionally, pE199L and pE248R, located in the inner viral membrane, are critical for membrane fusion, a necessary step for viral entry into host cells. Another important player is CD2v, a type I transmembrane protein involved in the infection process. In this research, to develop subunit vaccines against ASFV, we focused on cloning the genes encoding these five proteins—p49, p72, pE199L, pE248R, and CD2v—into the pPIC9K plasmid for expression in the Pichia pastoris GS115 yeast strain. The viral genomic DNA was extracted from blood samples of infected pigs, and the genes encoding the five proteins were successfully amplified using Phusion PCR. The PCR products of each gene were then digested with EcoRI and NotI restriction enzymes and ligated into the pPIC9K plasmid. After that, we transformed the recombinant plasmids into Escherichia coli DH5α for amplification and purification. The plasmids were subsequently linearized with SalI and introduced into P. pastoris GS115 through electroporation. The selection of appropriate media and PCR analysis of the genomic DNA confirmed the successful generation of five recombinant P. pastoris GS115 strains. This work paves the way for the development of a recombinant protein vaccine against ASF by using the Pichia pastoris GS115 in the future.

A liquid chromatography-tandem mass spectrometry method to determine total matrine residues in edible porcine tissues and its application in a tissue residue depletion study

Matrine (MT), a phytochemical with various anti-infective activities, is expected to become a therapeutic agent against bacterial, viral, and parasitic infections in pigs. However, food safety risks associated with MT have not yet been evaluated. In the present study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine total MT residues in edible porcine tissues. The tissue samples were hydrolyzed with trypsin to release non-extractable MT. The hydrolysates were extracted with a trichloroacetic acid solution. Tissue extracts were purified using solid-phase extraction and subjected to LC-MS/MS. The method was fully validated according to the United States Food and Drug Administration (USFDA) guidelines and applied to investigate the tissue residue depletion of MT in pigs. Significant accumulation of MT was observed in edible porcine tissues. Notably, MT showed no persistent tissue residue in this study and could be accepted as a safe anti-infective agent in pigs.

Antiviral effect of pinostrobin, a bioactive constituent of Boesenbergia rotunda, against porcine epidemic diarrhea virus.

Global swine industry has long been severely affected by the periodic outbreaks of porcine epidemic diarrhea (PED), a deadly infectious disease in piglets caused by the porcine epidemic diarrhea virus (PEDV). Currently, available vaccines and antiviral drugs could not provide effective prevention and treatment of PEDV infection in pigs. In this study, Boesenbergia rotunda (B. rotunda) extract and its major bioactive flavonoid, pinostrobin, were demonstrated to exhibit remarkable anti-PEDV activities with EC50 values of 0.33±0.02μg/ml and 2.71±0.12μM, and selectivity indices (SI) of 11.93 and>184.55, respectively. Results based on a time-of-addition assay showed that pinostrobin blocked PEDV infection mainly at the early stages of infection. More specifically, pinostrobin reduced cell-cell fusion mediated by the viral spike protein, suggesting that the compound may target the virus fusion step. We also synthesized pinostrobin derivatives and explored the impact of pinostrobin structural features to the observed anti-PEDV activity. Results indicated the importance of the hydroxyl group and substituent on the phenyl ring. In summary, this study highlights the potential of B. rotunda extract and its bioactive compound, pinostrobin, as candidates for the development of antiviral drugs to more effectively control PEDV infection.

Naturally occurring influenza reassortment in pigs facilitates the emergence of intrahost virus subpopulations with distinct genotypes and replicative fitness.

Pigs play a crucial role in driving influenza A virus (IAV) diversification and evolution by reassorting the viruses originating from different hosts. Despite IAV reassortment and diversity being well documented in pig populations at different scales (e.g., farm, region, country), limited field research has explored the extent of reassortment happening at the single pig level and how that contributes to the overall genetic and biological variation observed in populations. We provide initial information on levels of reassortment happening at the single pig level in naturally infected pigs, and that particular pigs can shed a plethora of distinct genotypes, with certain genotypes having distinct replicative fitness on swine and human respiratory tracts, which preserves the potential for IAV long-term evolution and facilitates the emergence of zoonotic/pandemic-capable reassortants.

High-throughput screening for respiratory pathogens within pigs in Denmark; analysis of circulating porcine respiratory coronaviruses and their association with other pathogens

Porcine respiratory coronavirus (PRCV) typically causes subclinical or mild respiratory infections in pigs, but may lead to more severe disease with other factors. PRCV infection in Denmark was initially detected in 1984, but data are lacking about its current prevalence and diversity. Antibodies against PRCV were detected in about 75 % of recent pig sera from Denmark. In addition, pig nasal swab samples were screened for PRCV and 12 other respiratory pathogens using a high-throughput RT-qPCR system. All targeted pathogens were detected but at different prevalences. Significant associations were found between the presence of PRCV and certain other pathogens. From PRCV positive samples, partial spike gene sequences and complete nucleocapsid coding sequences were determined. In phylogenetic analyses, these PRCVs clustered with earlier European PRCVs and were distinct from transmissible gastroenteritis virus. We conclude that PRCV is widespread within the pig population in Denmark. Further studies on the significance of PRCV are warranted.

Development of point-of-care immunodiagnostic test for Taenia solium cysticercosis in pigs

Cysticercosis, caused by the larval stage of Taenia solium, is a major zoonotic parasitic disease with a global impact, heightened by immigration and transboundary activities. Recognizing its importance, the World Health Organization has prioritized eliminating taeniasis/cysticercosis by 2030, designating it as a priority neglected tropical disease (NTD). Accurate diagnosis is essential for effective disease control, yet traditional methods often fall short due to low sensitivity of the test and the need for specialized laboratory setups. This study focuses on developing a recombinant Ag2 (rAg2)-based dot blot assay to detect T. solium cysticercosis in pigs. The rAg2 antigen was successfully expressed and purified, with immunoreactivity confirmed against sera from naturally infected pigs. The dot blot test evaluated against serological gold-standard enzyme-linked immunotransfer blot (EITB) assay, demonstrated preliminary laboratory sensitivity (89 %, 95 % CI: 68.6–97.1) and specificity (100 %, 95 % CI: 97.6 - 100), highlighting its potential as a reliable alternative diagnostic method. Cost-effectiveness and minimal laboratory requirements make it particularly suited for field use in resource-limited settings.

Vimentin as a universal receptor for pseudorabies virus infection in pig and human cells

Pseudorabies virus (PRV), known to infect pigs and found in various species, including humans, shows zoonotic potential. This study identified vimentin (VIM), a highly conserved intermediate filament protein expressed in multiple mammalian species and tissues, as a universal receptor for PRV infections in human and porcine cells. The adsorption of PRV is positively correlated with the level of VIM expressed in different cells. Overexpression and knockdown of VIM significantly increase and decrease PRV adsorption in cells, respectively. Dot blot assay and VOPBA showed that purified VIM can directly bind to PRV virions efficiently in a dose-dependent manner. PRV and VIM are co-localized at the cell membrane of PRV-infected cells. Moreover, PRV mainly binds to host VIM via its conserved amino acid residues in PRV gD (W98, G162, Y164, C205) and gH (C439) and the Rod domain (residues 96–404) of VIM. In addition, regulating the expression of VIM also influences the entry, replication, and release of PRV, which has a similar result to the adsorption. These results demonstrate that VIM, as a universal receptor, can facilitate PRV infection in multiple stages in human and porcine cells, highlighting the zoonosis characteristics of PRV and the need for more attention.

Enhanced pathogenicity and synergistic effects of co-infection with bovine viral diarrhea virus 1 and HoBi-like virus in cattle and guinea pigs.

The Bovine Viral Diarrhea Virus 1 (BVDV1) and HoBi-like virus (BVDV3), both within the same genus, share genomic homology and exhibit low antigenic cross-reactivity despite presenting similar clinical manifestations. In 2021, a bovine respiratory disease complex (BRDC) outbreak on two cattle farms in the Inner Mongolia Autonomous Region of China resulted in ten fatalities. Metagenomic and metatranscriptomic analyses were used to identify viral agents, including a co-infection case. A genetic evolution analysis assessed the relationships with related strains. Experimental infections in guinea pigs and calves evaluated the pathogenicity of the viruses. Phylogenetic analysis of the BVDV3 isolate IM2201 revealed close relatedness to Brazilian strains, with 97.06% nucleotide homology to the highly virulent strain SV478/07. Experimental co-infection in guinea pigs resulted in more severe clinical signs, including fever, cough, diarrhea, and significant pathological changes, and led to a higher mortality rate (40%) compared to no mortality from single-virus infections with BVDV1 or BVDV3. Similarly, co-infected cattle exhibited more severe clinical signs, including bloody diarrhea and rectal temperatures exceeding 40°C, along with persistent viremia and nasal viral shedding from 7 to 21 days post-infection. Blood analysis revealed significant reductions in white blood cell counts, particularly in co-infected cattle. This study highlights the enhanced pathogenicity and synergistic effects of BVDV1 and BVDV3 co-infection, exacerbating disease severity.

Quantifying the impact of vaccination on transmission and diversity of influenza A variants in pigs.

Global evolutionary dynamics of influenza A virus (IAV) are fundamentally driven by the extent of virus diversity generated, transmitted, and shaped in individual hosts. How vaccination affects the degree of IAV genetic diversity that can be transmitted and expanded in pigs is unknown. To evaluate the effect of vaccination on the transmission of genetically distinct IAV variants and their diversity after transmission in pigs, we examined the whole genome of IAV recovered from the nasal cavities of pigs vaccinated with different influenza immunization regimens after being infected simultaneously by H1N1 and H3N2 IAVs using a seeder pig model. We found that the seeder pigs harbored more diversified virus populations than the contact pigs. Among contact pigs, H3N2 and H1N1 viruses recovered from pigs vaccinated with a single dose of an unmatched modified live vaccine generally accumulated more extensive genetic mutations than non-vaccinated pigs. Furthermore, the non-sterilizing immunity elicited by the single-dose-modified live vaccine may have exerted positive selection on H1 antigenic regions as we detected significantly higher nonsynonymous but lower synonymous evolutionary rates in H1 antigenic regions than non-antigenic regions. In addition, we observed that the vaccinated pigs shared significantly less proportion of H3N2 variants with seeder pigs than unvaccinated pigs. These results indicated that vaccination might reduce the impact of transmitted influenza variants on the overall diversity of IAV populations harbored in recipient pigs and that within-host genetic selection of IAV is more likely to occur in pigs vaccinated with improperly matched vaccines.IMPORTANCEUnderstanding how vaccination shapes the diversity of influenza variants that transmit and propagate among pigs is essential for designing effective IAV surveillance and control programs. Current knowledge about the transmission of IAV variants has primarily been explored in humans during natural infection. However, how immunity elicited by improperly matched vaccines affects the degree of IAV genetic diversity that can be transmitted and expanded in pigs at the whole-genome level is unknown. We analyzed IAV sequences from samples collected daily from experimentally infected pigs vaccinated with various protocols in a field-represented IAV co-infection model. We found that vaccine-induced non-sterilizing immunity might promote genetic variation on the IAV genome and drive positive selection at antigenic sites during infection. In addition, a smaller proportion of H3N2 viral variants were shared between seeder pigs and vaccinated pigs, suggesting the influence of vaccination on shaping the virus genomic diversity in recipient pigs during the transmission events.

Characterization of a Mycoplasma hyopneumoniae aerosol infection model in pigs

The purpose of the present study was to develop and characterize an experimental aerosol model for Mycoplasma hyopneumoniae (M. hyopneumoniae) infection and respiratory disease in pigs. The experiment was carried out to determine the pathogenicity, colonization, mucosal immune response, and clinical course of disease of dose-controlled aerosols of M. hyopneumoniae. Four groups of three M. hyopneumoniae-free gilts each were individually exposed to aerosols of diluted lung homogenate containing M. hyopneumoniae strain 232 in a chamber. Each group was exposed to different doses of viable organisms (105 to 106 color changing units/mL during 15–20 or 30–35 min in two consecutive days). Nasal, laryngeal, and deep-tracheal secretions were collected from each gilt at 0, 7, 14, 21, and 28 days post-exposure (dpe). Blood samples were collected at 0 and 28 dpe. At necropsy, lung lesions were assessed, and bronchial secretions and bronchoalveolar lavage fluid (BALF) were collected from each lung set. Blood was used to assess seroconversion by means of an indirect ELISA, while BALF, deep-tracheal and nasal secretions were tested by modifying the ELISA to evaluate mucosal IgG and IgA production. Nasal, laryngeal, deep-tracheal, and bronchial secretions were tested by real-time PCR to evaluate bacterial load. Gilts became infected irrespective of the infectious dose, as determined by M. hyopneumoniae detection in deep-tracheal secretions from all gilts at 7 dpe. A specific local humoral immune response starting at 14 dpe was detected in all gilts. While all experimental groups presented gilts with some extent of mycoplasmal pneumonia, only the exposure of gilts to high-dose aerosols consistently reproduced typical clinical signs and severe lung lesions. These results showed that the reproduction of mycoplasmal pneumonia by means of infectious aerosols can be successfully achieved at experimental level, making this model a valuable alternative to evaluate preventive and treatment measures against M. hyopneumoniae.

CD4+ but not CD8+ T cells are required for protection against severe guinea pig cytomegalovirus infections.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus and the leading cause of infectious disease related birth defects worldwide. How the immune response modulates the risk of intrauterine transmission of HCMV after maternal infection remains poorly understood. Maternal T cells likely play a critical role in preventing infection at the maternal-fetal interface and limiting spread across the placenta, but concerns exist that immune responses to infection may also cause placental dysfunction and adverse pregnancy outcomes. This study investigated the role of CD4+ and CD8+ T cells in a guinea pig model of primary cytomegalovirus infection. Monoclonal antibodies specific to guinea pig CD4 and CD8 were used to deplete T cells in non-pregnant and in pregnant guinea pigs after mid-gestation. CD4+ T cell depletion increased the severity of illness, caused significantly elevated viral loads, and increased the rate of congenital guinea pig cytomegalovirus (GPCMV) infection relative to animals treated with control antibody. CD8+ T cell depletion was comparably well tolerated and did not significantly affect the weight of infected guinea pigs or viral loads in their blood or tissue. However, significantly more viral genomes and transcripts were detected in the placenta and decidua of CD8+ T cell depleted dams post-infection. This study corroborates earlier findings made in nonhuman primates that maternal CD4+ T cells play a critical role in limiting the severity of primary CMV infection during pregnancy while also revealing that other innate and adaptive immune responses can compensate for an absent CD8+ T cell response in α-CD8-treated guinea pigs.