Fructose-asparagine is a primary nutrient during growth of Salmonella in the inflamed intestine.

Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that Salmonella encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for Salmonella fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10-/- mice). The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1- SPI2- or ttrA mutants, respectively). The severe fitness defect of a Salmonella fra mutant suggests that F-Asn is the primary nutrient utilized by Salmonella in the inflamed intestine and that this system provides a valuable target for novel therapies.

mRNA cap methylation influences pathogenesis of vesicular stomatitis virus in vivo.

UNLABELLED: One role of mRNA cap guanine-N-7 (G-N-7) methylation is to facilitate the efficient translation of mRNA. The role of mRNA cap ribose 2'-O methylation is enigmatic, although recent work has implicated this as a signature to avoid detection of RNA by the innate immune system (S. Daffis, K. J. Szretter, J. Schriewer, J. Q. Li, S. Youn, J. Errett, T. Y. Lin, S. Schneller, R. Zust, H. P. Dong, V. Thiel, G. C. Sen, V. Fensterl, W. B. Klimstra, T. C. Pierson, R. M. Buller, M. Gale, P. Y. Shi, M. S. Diamond, Nature 468:452-456, 2010, doi:10.1038/nature09489). Working with vesicular stomatitis virus (VSV), we previously showed that a panel of recombinant VSVs carrying mutations at a predicted methyltransferase catalytic site (rVSV-K1651A, -D1762A, and -E1833Q) or S-adenosylmethionine (SAM) binding site (rVSV-G1670A, -G1672A, and -G4A) were defective in cap methylation and were also attenuated for growth in cell culture. Here, we analyzed the virulence of these recombinants in mice. We found that rVSV-K1651A, -D1762A, and -E1833Q, which are defective in both G-N-7 and 2'-O methylation, were highly attenuated in mice. All three viruses elicited a high level of neutralizing antibody and provided full protection against challenge with the virulent VSV. In contrast, mice inoculated with rVSV-G1670A and -G1672A, which are defective only in G-N-7 methylation, were attenuated in vivo yet retained a low level of virulence. rVSV-G4A, which is completely defective in both G-N-7 and 2'-O methylation, also exhibited low virulence in mice despite the fact that productive viral replication was not detected in lung and brain. Taken together, our results suggest that abrogation of viral mRNA cap methylation can serve as an approach to attenuate VSV, and perhaps other nonsegmented negative-strand RNA viruses, for potential application as vaccines and viral vectors. IMPORTANCE: Nonsegmented negative-sense (NNS) RNA viruses include a wide range of significant human, animal, and plant pathogens. For many of these viruses, there are no vaccines or antiviral drugs available. mRNA cap methylation is essential for mRNA stability and efficient translation. Our current understanding of mRNA modifications of NNS RNA viruses comes largely from studies of vesicular stomatitis virus (VSV). In this study, we showed that recombinant VSVs (rVSVs) defective in mRNA cap methylation were attenuated in vitro and in vivo. In addition, these methyltransferase (MTase)-defective rVSVs triggered high levels of antibody responses and provided complete protection against VSV infection. Thus, this study will not only contribute to our understanding of the role of mRNA cap MTase in viral pathogenesis but also facilitate the development of new live attenuated vaccines for VSV, and perhaps other NNS RNA viruses, by inhibiting viral mRNA cap methylation.

A Gnotobiotic Pig Model for Determining Human Norovirus Inactivation by High-Pressure Processing.

Human norovirus (NoV) is responsible for over 90% of outbreaks of acute nonbacterial gastroenteritis worldwide and accounts for 60% of cases of foodborne illness in the United States. Currently, the infectivity of human NoVs is poorly understood due to the lack of a cell culture system. In this study, we determined the survival of a human NoV genogroup II, genotype 4 (GII.4) strain in seeded oyster homogenates after high-pressure processing (HPP) using a novel receptor binding assay and a gnotobiotic pig model. Pressure conditions of 350 MPa at 0°C for 2 min led to a 3.7-log10 reduction in the number of viral RNA copies in oysters, as measured by the porcine gastric mucin-conjugated magnetic bead (PGM-MB) binding assay and real-time RT-PCR, whereas pressure conditions of 350 MPa at 35°C for 2 min achieved only a 1-log10 reduction in the number of RNA copies. Newborn gnotobiotic piglets orally fed oyster homogenate treated at 350 MPa and 0°C for 2 min did not have viral RNA shedding in feces, histologic lesions, or viral replication in the small intestine. In contrast, gnotobiotic piglets fed oysters treated at 350 MPa and 35°C for 2 min had high levels of viral shedding in feces and exhibited significant histologic lesions and viral replication in the small intestine. Collectively, these data demonstrate that (i) human NoV survival estimated by an in vitro PGM-MB virus binding assay is consistent with the infectivity determined by an in vivo gnotobiotic piglet model and (ii) HPP is capable of inactivating a human NoV GII.4 strain at commercially acceptable pressure levels.

Antibody responses to Bordetella bronchiseptica in vaccinated and infected dogs.

Bordetella bronchiseptica (Bb) whole cell bacterins have been replaced with acelluar vaccines. We evaluated the response to the acellular Bb vaccines in Bb-seropositive commingled laboratory beagles and client-owned dogs with various lifestyles and vaccination histories. A single parenteral dose of the acellular Bb vaccine resulted in consistent anamnestic IgG, and to a lesser, but notable extent, IgA, Bb-reactive antibody responses in the seropositive beagles. Associated with the increase in antibodies measured by enzyme-linked immunosorbent assay (ELISA) was an increase in the complement (C)-dependent IgG antibody mediated bactericidal effect on Bb in vitro. Antibody responses in client-owned dogs were more variable and were dependent upon the vaccination history and serological evidence of previous Bb exposure. Antibodies from vaccinated dogs recognized several Bb proteins, notably P68 (pertactin) and P220 (fimbrial hemagglutinin), the response to which has been shown to be disease-sparing in Bp infections. These antibody responses were similar to those in experimentally infected dogs and in dogs that had received a widely used whole cell bacterin.

Réponses des anticorps àBordetella bronchisepticachez des chiens vaccinés et infectés. Des bactérines de Bordetella bronchiseptica (Bb) ont été remplacées par des vaccins acellulaires. Nous avons évalué la réponse aux vaccins Bb acellulaires chez un groupe de Beagles de laboratoire séropositifs pour le Bb et des chiens de clients ayant divers styles de vie et antécédents de vaccination. Une seule dose parentérale du vaccin Bb acellulaire s'est traduite par une réponse IgG anamnestique uniforme, et à degré inférieur mais significatif, des réponses IgA et des anticorps réactifs à Bb chez les Beagles séropositifs. Une hausse des anticorps mesurés par ELISA a été accompagnée d'une augmentation de l'effet bactéricide atténué des anticorps dépendants IgG du complément (C) sur Bb in vitro. Les réponses des anticorps chez les chiens appartenant à des clients étaient plus variables et dépendaient des antécédents de vaccination et des preuves sérologiques d'une exposition antérieure à Bb. Les anticorps de chiens vaccinés reconnaissaient plusieurs protéines Bb, notamment P68 (pertactine) et P220 (hémagglutinine fimbriale), dont la réponse a été démontrée comme une protection contre la maladie lors d'une infection par Bb. Ces réponses des anticorps étaient semblables à celles des chiens infectés par expérimentation et à celles des chiens qui avaient reçu des bactérines à bacilles entiers généralement utilisés.(Traduit par Isabelle Vallières).

Swine influenza H1N1 virus induces acute inflammatory immune responses in pig lungs: a potential animal model for human H1N1 influenza virus.

Pigs are capable of generating reassortant influenza viruses of pandemic potential, as both the avian and mammalian influenza viruses can infect pig epithelial cells in the respiratory tract. The source of the current influenza pandemic is H1N1 influenza A virus, possibly of swine origin. This study was conducted to understand better the pathogenesis of H1N1 influenza virus and associated host mucosal immune responses during acute infection in humans. Therefore, we chose a H1N1 swine influenza virus, Sw/OH/24366/07 (SwIV), which has a history of transmission to humans. Clinically, inoculated pigs had nasal discharge and fever and shed virus through nasal secretions. Like pandemic H1N1, SwIV also replicated extensively in both the upper and lower respiratory tracts, and lung lesions were typical of H1N1 infection. We detected innate, proinflammatory, Th1, Th2, and Th3 cytokines, as well as SwIV-specific IgA antibody in lungs of the virus-inoculated pigs. Production of IFN-γ by lymphocytes of the tracheobronchial lymph nodes was also detected. Higher frequencies of cytotoxic T lymphocytes, γδ T cells, dendritic cells, activated T cells, and CD4+ and CD8+ T cells were detected in SwIV-infected pig lungs. Concomitantly, higher frequencies of the immunosuppressive T regulatory cells were also detected in the virus-infected pig lungs. The findings of this study have relevance to pathogenesis of the pandemic H1N1 influenza virus in humans; thus, pigs may serve as a useful animal model to design and test effective mucosal vaccines and therapeutics against influenza virus.

Immunity and Protective Efficacy of Mannose Conjugated Chitosan-Based Influenza Nanovaccine in Maternal Antibody Positive Pigs.

Parenteral administration of killed/inactivated swine influenza A virus (SwIAV) vaccine in weaned piglets provides variable levels of immunity due to the presence of preexisting virus specific maternal derived antibodies (MDA). To overcome the effect of MDA on SwIAV vaccine in piglets, we developed an intranasal deliverable killed SwIAV antigen (KAg) encapsulated chitosan nanoparticles called chitosan-based NPs encapsulating KAg (CS NPs-KAg) vaccine. Further, to target the candidate vaccine to dendritic cells and macrophages which express mannose receptor, we conjugated mannose to chitosan (mCS) and formulated KAg encapsulated mCS nanoparticles called mannosylated chitosan-based NPs encapsulating KAg (mCS NPs-KAg) vaccine. In MDA-positive piglets, prime-boost intranasal inoculation of mCS NPs-KAg vaccine elicited enhanced homologous (H1N2-OH10), heterologous (H1N1-OH7), and heterosubtypic (H3N2-OH4) influenza virus-specific secretory IgA (sIgA) antibody response in nasal passage compared to CS NPs-KAg vaccinates. In vaccinated upon challenged with a heterologous SwIAV H1N1, both mCS NPs-KAg and CS NPs-KAg vaccinates augmented H1N2-OH10, H1N1-OH7, and H3N2-OH4 virus-specific sIgA antibody responses in nasal swab, lung lysate, and bronchoalveolar lavage (BAL) fluid; and IgG antibody levels in lung lysate and BAL fluid samples. Whereas, the multivalent commercial inactivated SwIAV vaccine delivered intramuscularly increased serum IgG antibody response. In mCS NPs-KAg and CS NPs-KAg vaccinates increased H1N2-OH10 but not H1N1-OH7 and H3N2-OH4-specific serum hemagglutination inhibition titers were observed. Additionally, mCS NPs-KAg vaccine increased specific recall lymphocyte proliferation and cytokines IL-4, IL-10, and IFNγ gene expression compared to CS NPs-KAg and commercial SwIAV vaccinates in tracheobronchial lymph nodes. Consistent with the immune response both mCS NPs-KAg and CS NPs-KAg vaccinates cleared the challenge H1N1-OH7 virus load in upper and lower respiratory tract more efficiently when compared to commercial vaccine. The virus clearance was associated with reduced gross lung lesions. Overall, mCS NP-KAg vaccine intranasal immunization in MDA-positive pigs induced a robust cross-reactive immunity and offered protection against influenza virus.

Poly(I:C) augments inactivated influenza virus-chitosan nanovaccine induced cell mediated immune response in pigs vaccinated intranasally.

To improve the innate and adaptive immune responses elicited by a killed/inactivated swine influenza virus antigen (KAg)-loaded chitosan nanoparticles (CS NPs-KAg), we used the adjuvant, poly(I:C). The formulated CS NPs-KAg and CS NPs-poly(I:C) had a net surface charge of +30.7 mV and +25.1 mV, respectively. The CS NPs-KAg was coadministered with CS NPs-poly(I:C) (chitosan nanovaccine) as intranasal mist. Vaccinations enhanced homologous (H1N2-OH10) and heterologous (H1N1-OH7) hemagglutination inhibition (HI) titers in both vaccinated and virus-challenged animals compared to the control soluble poly(I:C) vaccinated pigs. In addition, the chitosan nanovaccine induced the proliferation of antigen-specific IFNγ secreting T-helper/memory and γδ T cells compared to control poly(I:C) group; and an increased Th1 (IFNγ, IL-6 and IL-2) and Th2 (IL-10 and IL-13) cytokines mRNA expression in the tracheobronchial lymph nodes compared to lymphoid tissues obtained from pigs given commercial influenza vaccine. The virus load in nasal passages and microscopic lung lesions were partially reduced by both chitosan nanovaccine and commercial vaccine. The HA gene homology between the vaccine and challenge viruses indicated that the chitosan nanovaccine induced a cross-protective immune response. In conclusion, coadministration of CS NPs-poly(I:C) with CS NPs-KAg augmented the cross-reactive specific HI titers and the cell-mediated immune responses in pigs.

Can Unconventional Immunomodulatory Agents Help Alleviate COVID-19 Symptoms and Severity?

Severe acute respiratory syndrome coronavirus 2 (SARS coronavirus 2, or SARS-CoV-2) is the cause of the respiratory infection known as COVID-19. From an immunopathological standpoint, coronaviruses such as SARS-CoV-2 induce increased levels of a variety of T-helper 1 (Th1) and inflammatory cytokines and chemokines, including interleukin-1 (IL-1), IL-6, CCL2 protein, and CXCL10 protein. In the absence of proven antiviral agents or an effective vaccine, substances with immunomodulatory activity may be able to inhibit inflammatory and Th1 cytokines and/or yield an anti-inflammatory and/or Th2 immune response to counteract COVID-19 symptoms and severity. This report briefly describes the following four unconventional but commercially accessible immunomodulatory agents that can be employed in clinical trials to evaluate their effectiveness at alleviating disease symptoms and severity: low-dose oral interferon alpha, microdose DNA, low-dose thimerosal, and phytocannabinoids.

A Nanoparticle-Poly(I:C) Combination Adjuvant Enhances the Breadth of the Immune Response to Inactivated Influenza Virus Vaccine in Pigs.

Intranasal vaccination elicits secretory IgA (SIgA) antibodies in the airways, which is required for cross-protection against influenza. To enhance the breadth of immunity induced by a killed swine influenza virus antigen (KAg) or conserved T cell and B cell peptides, we adsorbed the antigens together with the TLR3 agonist poly(I:C) electrostatically onto cationic alpha-D-glucan nanoparticles (Nano-11) resulting in Nano-11-KAg-poly(I:C) and Nano-11-peptides-poly(I:C) vaccines. In vitro, increased TNF-α and IL-1ß cytokine mRNA expression was observed in Nano-11-KAg-poly(I:C)-treated porcine monocyte-derived dendritic cells. Nano-11-KAg-poly(I:C), but not Nano-11-peptides-poly(I:C), delivered intranasally in pigs induced high levels of cross-reactive virus-specific SIgA antibodies secretion in the nasal passage and lungs compared to a multivalent commercial influenza virus vaccine administered intramuscularly. The commercial and Nano-11-KAg-poly(I:C) vaccinations increased the frequency of IFNγ secreting T cells. The poly(I:C) adjuvanted Nano-11-based vaccines increased various cytokine mRNA expressions in lymph nodes compared to the commercial vaccine. In addition, Nano-11-KAg-poly(I:C) vaccine elicited high levels of virus neutralizing antibodies in bronchoalveolar lavage fluid. Microscopic lung lesions and challenge virus load were partially reduced in poly(I:C) adjuvanted Nano-11 and commercial influenza vaccinates. In conclusion, compared to our earlier study with Nano-11-KAg vaccine, addition of poly(I:C) to the formulation improved cross-protective antibody and cytokine response.

Development and validation of a SYBR green real-time PCR for the quantification of porcine circovirus type 2 in serum, buffy coat, feces, and multiple tissues.

The emergence of multiple genotypes of PCV2, as demonstrated by phylogenetic analysis of whole genome or capsid sequences, makes it necessary to have quantitative diagnostic assays that perform equally well on all strains. The objectives of this study were to develop and validate a novel real-time polymerase chain reaction (PCR) assay targeting the highly conserved rep gene (ORF1) and investigate the effects of diagnostic specimen choice on its performance. The assay was tested in naturally infected conventional pigs, experimentally infected gnotobiotic pigs, and plasmid-spiked negative serum, lung tissue, and feces and found to have a linear detection range of 2.2x10(3) to 2.2x10(10) copies of PCV2 per mL. The assay successfully detected and quantified PCV2 DNA in serum, buffy coat, feces, and multiple lymphoid (bronchial, mesenteric, and superficial inguinal lymph nodes; thymus; tonsil; ileal Peyer's patches; and spleen), and non-lymphoid (myocardium; lung; kidney; liver; and gluteal muscle) tissues from naturally infected pigs. Across all tissues and sera of naturally infected pigs, the mean PCV2 concentration was 3.0logs higher in wasting versus non-wasting pigs. PCV2 concentration measured by tissue culture and immunohistochemical staining in homogenized liver samples of experimentally infected gnotobiotic pigs were compared to the concentrations estimated by quantitative PCR. Similar trends were noted with increasing PCV2 concentration detected in subclinically infected to severely PMWS-affected pigs across all assays. Our diagnostic assay was developed with a conserved target sequence, and performed efficiently in quantification of PCV2 in a variety of tissues from naturally and experimentally infected pigs.

Amish (Rural) vs. non-Amish (Urban) Infant Fecal Microbiotas Are Highly Diverse and Their Transplantation Lead to Differences in Mucosal Immune Maturation in a Humanized Germfree Piglet Model.

The gut microbiome plays an important role in the immune system development, maintenance of normal health status, and in disease progression. In this study, we comparatively examined the fecal microbiomes of Amish (rural) and non-Amish (urban) infants and investigated how they could affect the mucosal immune maturation in germ-free piglets that were inoculated with the two types of infant fecal microbiota (IFM). Differences in microbiome diversity and structure were noted between the two types of fecal microbiotas. The fecal microbiota of the non-Amish (urban) infants had a greater relative abundance of Actinobacteria and Bacteroidetes phyla, while that of the Amish (rural) counterparts was dominated by Firmicutes. Amish infants had greater species richness compared with the non-Amish infants' microbiota. The fecal microbiotas of the Amish and the non-Amish infants were successfully transplanted into germ-free piglets, and the diversity and structure of the microbiota in the transplanted piglets remained similar at phylum level but not at the genus level. Principal coordinates analysis (PCoA) based on Weighted-UniFrac distance revealed distinct microbiota structure in the intestines of the transplanted piglets. Shotgun metagenomic analysis also revealed clear differences in functional diversity of fecal microbiome between Amish and non-Amish donors as well as microbiota transplanted piglets. Specific functional features were enriched in either of the microbiota transplanted piglet groups directly corresponding to the predominance of certain bacterial populations in their gut environment. Some of the colonized bacterial genera were correlated with the frequency of important lymphoid and myeloid immune cells in the ileal submucosa and mesenteric lymph nodes (MLN), both important for mucosal immune maturation. Overall, this study demonstrated that transplantation of diverse IFM into germ-free piglets largely recapitulates the differences in gut microbiota structure between rural (Amish) and urban (non-Amish) infants. Thus, fecal microbiota transplantation to germ-free piglets could be a useful large animal model system for elucidating the impact of gut microbiota on the mucosal immune system development. Future studies can focus on determining the additional advantages of the pig model over the rodent model.

Porcine circovirus-2 DNA concentration distinguishes wasting from nonwasting pigs and is correlated with lesion distribution, severity, and nucleocapsid staining intensity.

The emergence of severe porcine circoviral disease in North America is associated with Porcine circovirus-2 genotype b (PCV-2b), which has led to speculation that PCV-2b is more virulent than PCV-2a. The objectives of this study were to 1) correlate the PCV-2 DNA concentration and lesions in wasting (WST) and age-matched healthy (HLTH) pigs from 2 clinically affected farms, and unaffected (UNFCT) pigs from a farm with no prior clinical or diagnostic history of PCVD; and 2) to determine the initial estimates of sensitivity and specificity of PCV-2 quantitative polymerase chain reaction (qPCR). PCV-2b was confirmed in all 3 farms. Compared with HLTH pigs, WST pigs demonstrated significantly more prevalent thymic atrophy, failure of normal pulmonary collapse, and ascites (P < 0.017 for all). The HLTH and UNFCT pigs had significantly more pronounced lymphoid germinal centers and proliferative paracortical T-dependent zones, compared with WST pigs (P < 0.017). Across all tissues, PCV-2 DNA concentrations were significantly higher in WST compared with HLTH and UNFCT pigs (P < 0.017 for all). The PCV-2 DNA concentrations were strongly correlated with PCV-2 nucleocapsid staining intensity in lymph node, spleen, Peyer's patches, lung, liver, and kidney (0.60 < or = r < or = 0.84). In the current study, the PCV-2 DNA log10 cutoff concentrations best able to distinguish WST from HLTH and UNFCT pigs were between 7.0 and 8.0 per gram for tissues, and between 4.0 and 5.0 per milliliter for sera. The presence of PCV-2b in UNFCT pigs is evidence that PCV-2b by itself is not sufficient to induce severe disease.

Effect of coinfection with genogroup 1 porcine torque teno virus on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gnotobiotic pigs.

OBJECTIVE: To determine whether genogroup 1 porcine torque teno virus (g1-TTV) can potentiate clinical disease associated with porcine circovirus type 2 (PCV2). SAMPLE POPULATION: 33 gnotobiotic baby pigs. PROCEDURES: Pigs were allocated into 7 groups: group A, 5 uninoculated control pigs from 3 litters; group B, 4 pigs oronasally inoculated with PCV2 alone; group C, 4 pigs inoculated IP with first-passage g1-TTV alone; group D, 4 pigs inoculated IP with fourth-passage g1-TTV alone; group E, 6 pigs inoculated IP with first-passage g1-TTV and then oronasally inoculated with PCV2 7 days later; group F, 6 pigs inoculated IP with fourth-passage g1-TTV and then inoculated oronasally with PCV2 7 days later; and group G, 4 pigs inoculated oro-nasally with PCV2 and then inoculated IP with fourth-passage g1-TTV 7 days later. RESULTS: 6 of 12 pigs inoculated with g1-TTV prior to PCV2 developed acute onset of postweaning multisystemic wasting syndrome (PMWS). None of the pigs inoculated with g1-TTV alone or PCV2 alone or that were challenge exposed to g1-TTV after establishment of infection with PCV2 developed clinical illness. Uninoculated control pigs remained healthy. CONCLUSIONS AND CLINICAL RELEVANCE: These data implicated g1-TTV as another viral infection that facilitates PCV2-induced PMWS. This raises the possibility that torque teno viruses in swine may contribute to disease expression currently associated with only a single infectious agent.

Evaluation of the effects of porcine genogroup 1 torque teno virus in gnotobiotic swine.

OBJECTIVE: To determine whether porcine genogroup 1 torque teno virus (g1-TTV) can infect and cause disease in gnotobiotic swine. SAMPLE POPULATION: 20 conventional baby pigs and 46 gnotobiotic baby pigs. PROCEDURES: Porcine g1-TTV was transmitted from conventional swine to gnotobiotic pigs via pooled leukocyte-rich plasmas (n=18) that had positive results for g1-TTV DNA. Bone marrow-liver homogenates that had positive results for torque teno virus (TTV) were used in 4 serial passages in gnotobiotic pigs (2 pigs/passage). A pathogenesis experiment was conducted with in vivo passages of g1-TTV in various groups of gnotobiotic pigs. RESULTS: All g1-TTV inoculated pigs had no clinical signs but developed interstitial pneumonia, transient thymic atrophy, membranous glomerulonephropathy, and modest lymphocytic to histiocytic infiltrates in the liver after inoculation with the TTV-containing tissue homogenate; these changes were not detected in uninoculated control pigs or pigs injected with tissue homogenate devoid of TTV DNAs. In situ hybridization was used to identify g1-TTV DNAs in bone marrow mononuclear cells. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these data revealed that porcine g1-TTV was readily transmitted to TTV-naïve swine and that infection was associated with characteristic pathologic changes in gnotobiotic pigs inoculated with g1-TTV. Thus, g1-TTV could be an unrecognized pathogenic viral infectious agent of swine. This indicated a directly associated induction of lesions attributable to TTV infection in swine for a virus of the genus Anellovirus.

Evaluation of induction of porcine dermatitis and nephropathy syndrome in gnotobiotic pigs with negative results for porcine circovirus type 2.

OBJECTIVE: To determine whether porcine dermatitis and nephropathy syndrome (PDNS) could be experimentally induced in gnotobiotic swine. SAMPLE POPULATION: Plasma samples from 27 sows and 20 conventional weaned piglets were obtained, and 30 gnotobiotic pigs were used in experiments. PROCEDURES: 3 experiments were conducted. Groups of 3-day-old gnotobiotic pigs were inoculated with pooled plasma samples obtained from healthy feeder pigs in a herd that was in the initial phases of an outbreak of respiratory disease; gross and histologic lesions of PDNS were detected in the inoculated pigs. In a second experiment, 2- and 3-day-old gnotobiotic pigs were inoculated with porcine reproductive respiratory syndrome virus (PRRSV) and with PRRSV-negative tissue homogenate containing genogroup 1 torque teno virus (g1-TTV). Lesions of PDNS were detected. RESULTS: Pigs inoculated with pooled plasma or the combination of tissue-culture-origin PRRSV and g1-TTV tissue homogenate developed systemic hemostatic defects, bilaterally symmetric cutaneous hemorrhages, generalized edema, icterus, bilaterally symmetric renal cortical hemorrhage, dermal vasculitis with hemorrhage, and interstitial pneumonia consistent with a clinical and pathologic diagnosis of PDNS. The PRRSV RNAs and g1-TTV DNAs were detected in plasma; all pigs seroconverted to PRRSV, and all had negative results for porcine circovirus type 2 when tested by use of PCR assays. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggested that PDNS is a manifestation of disseminated intravascular coagulation in swine. For the experimental conditions reported here, combined infection with g1-TTV and PRRSV was implicated in the genesis of these lesions.

Evaluation of Mycoplasma hyopneumoniae bacterins for porcine torque teno virus DNAs.

OBJECTIVE: To determine whether commercial Mycoplasma hyopneumoniae bacterins sold for use in swine contain porcine torque teno virus (TTV). SAMPLE POPULATION: 22 commercially available M hyopneumoniae bacterins. PROCEDURES: Direct and nested PCR assays for genogroup-specific TTV DNAs were performed on serials of M hyopneumoniae bacterins by use of published and custom-designed primer pairs at 3 laboratories in North America and Europe. RESULTS: Of the 22 bacterins tested by use of direct and nested PCR assays, 7 of 9 from the United States, 2 of 5 from Canada, and 4 of 8 from Europe contained genogroup 1- and genogroup 2-TTV DNAs. In some bacterins, the TTV DNAs were readily detected by use of direct PCR assays. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these data indicated that many of the commercially available M hyopneumoniae bacterins were contaminated with TTV DNA. It is possible that some of these bacterins could inadvertently transmit porcine TTV infection to TTV-naïve swine.

Quantitative polymerase chain reaction for Porcine circovirus-2 in swine feces in a Porcine circovirus disease-affected commercial herd and a nonaffected commercial herd.

This study examined if pigs in a Porcine circovirus disease (PCVD)-affected herd (n = 100) had shed more Porcine circovirus-2 (PCV-2) in their feces than pigs in a PCVD-nonaffected herd (n = 101), and if differences in shedding among production stages within and between the herds existed. The PCV-2 shedding was quantified by real-time polymerase chain reaction. The highest median PCV-2 shedding was found in the nursery of the PCVD-affected herd and in the grower of the PCVD-nonaffected herd. The PCV-2 shedding was significantly higher in earlier stages (newly weaned, nursery, and pregrower) in the PCVD-affected herd (Wilcoxon rank sum; P < 0.001) compared with the PCVD-nonaffected herd. Porcine circovirus-2 DNA was not detected in a significant proportion of lactating sows (parity > or = 3) in the PCVD-nonaffected herd (Fisher's exact test; P = 0.001). The results of this study suggest there may be an association between the presence of PCV-2 in the feces of lactating sows and increased PCV-2 shedding in younger pigs.

Mucosal Immunity and Protective Efficacy of Intranasal Inactivated Influenza Vaccine Is Improved by Chitosan Nanoparticle Delivery in Pigs.

Annually, swine influenza A virus (SwIAV) causes severe economic loss to swine industry. Currently used inactivated SwIAV vaccines administered by intramuscular injection provide homologous protection, but limited heterologous protection against constantly evolving field viruses, attributable to the induction of inadequate levels of mucosal IgA and cellular immune responses in the respiratory tract. A novel vaccine delivery platform using mucoadhesive chitosan nanoparticles (CNPs) administered through intranasal (IN) route has the potential to elicit strong mucosal and systemic immune responses in pigs. In this study, we evaluated the immune responses and cross-protective efficacy of IN chitosan encapsulated inactivated SwIAV vaccine in pigs. Killed SwIAV H1N2 (δ-lineage) antigens (KAg) were encapsulated in chitosan polymer-based nanoparticles (CNPs-KAg). The candidate vaccine was administered twice IN as mist to nursery pigs. Vaccinates and controls were then challenged with a zoonotic and virulent heterologous SwIAV H1N1 (γ-lineage). Pigs vaccinated with CNPs-KAg exhibited an enhanced IgG serum antibody and mucosal secretory IgA antibody responses in nasal swabs, bronchoalveolar lavage (BAL) fluids, and lung lysates that were reactive against homologous (H1N2), heterologous (H1N1), and heterosubtypic (H3N2) influenza A virus strains. Prior to challenge, an increased frequency of cytotoxic T lymphocytes, antigen-specific lymphocyte proliferation, and recall IFN-γ secretion by restimulated peripheral blood mononuclear cells in CNPs-KAg compared to control KAg vaccinates were observed. In CNPs-KAg vaccinated pigs challenged with heterologous virus reduced severity of macroscopic and microscopic influenza-associated pulmonary lesions were observed. Importantly, the infectious SwIAV titers in nasal swabs [days post-challenge (DPC) 4] and BAL fluid (DPC 6) were significantly (p < 0.05) reduced in CNPs-KAg vaccinates but not in KAg vaccinates when compared to the unvaccinated challenge controls. As well, an increased frequency of T helper memory cells and increased levels of recall IFNγ secretion by tracheobronchial lymph nodes cells were observed. In summary, chitosan SwIAV nanovaccine delivered by IN route elicited strong cross-reactive mucosal IgA and cellular immune responses in the respiratory tract that resulted in a reduced nasal viral shedding and lung virus titers in pigs. Thus, chitosan-based influenza nanovaccine may be an ideal candidate vaccine for use in pigs, and pig is a useful animal model for preclinical testing of particulate IN human influenza vaccines.

Liposomal nanoparticle-based conserved peptide influenza vaccine and monosodium urate crystal adjuvant elicit protective immune response in pigs.

BACKGROUND: Influenza (flu) is a constant threat to humans and animals, and vaccination is one of the most effective ways to mitigate the disease. Due to incomplete protection induced by current flu vaccines, development of novel flu vaccine candidates is warranted to achieve greater efficacy against constantly evolving flu viruses. METHODS: In the present study, we used liposome nanoparticle (<200 nm diameter)-based subunit flu vaccine containing ten encapsulated highly conserved B and T cell epitope peptides to induce protective immune response against a zoonotic swine influenza A virus (SwIAV) H1N1 challenge infection in a pig model. Furthermore, we used monosodium urate (MSU) crystals as an adjuvant and co-administered the vaccine formulation as an intranasal mist to flu-free nursery pigs, twice at 3-week intervals. RESULTS: Liposome peptides flu vaccine delivered with MSU adjuvant improved the hemagglutination inhibition antibody titer and mucosal IgA response against the SwIAV challenge and also against two other highly genetically variant IAVs. Liposomal vaccines also enhanced the frequency of peptides and virus-specific T-helper/memory cells and IFN-γ response. The improved specific cellular and mucosal humoral immune responses in adjuvanted liposomal peptides flu vaccine partially protected pigs from flu-induced fever and pneumonic lesions, and reduced the nasal virus shedding and viral load in the lungs. CONCLUSION: Overall, our study shows great promise for using liposome and MSU adjuvant- based subunit flu vaccine through the intranasal route, and provides scope for future, pre-clinical investigations in a pig model for developing potent human intranasal subunit flu vaccines.

A novel method for the detection of porcine circovirus type 2 replicative double stranded viral DNA and nonreplicative single stranded viral DNA in tissue sections.

Porcine circovirus type 2 (PCV2), an economically important pathogen of swine, is the necessary cause of post weaning multisystemic wasting disease (PMWS); PCV2 infection is associated with porcine dermatitis and nephritis syndrome (PDNS). Current immunohistochemical (IHC) methodologies identify PCV2 antigens but are not capable of differentiating replicating virus from nonreplicating virion particles in tissue sections. In this paper, a combination of IHC using commercial monoclonal antibodies specific for single stranded (ss) and double stranded (ds) DNA and PCV2 specific in situ hybridization (ISH) was used to show the specificity of the former for PCV2 DNA in tissue sections from PCV2-infected gnotobiotic pigs. Cold-ethanol-fixed tissue sections were superior to formalin-fixed tissues for detection of PCV2 DNA, presumably due to the lack of protein cross-linking in the latter. These data demonstrate that conventional IHC detects PCV2 DNA forms in experimentally infected PCV2-positive gnotobiotic porcine tissue sections that are minimally compromised by either formalin fixation or the hybridization conditions needed for ISH.