Knock down of transforming growth factor beta improves expressions of co-stimulatory molecules, type I interferon-regulated genes, and pro-inflammatory cytokine in PRRSV-inoculated monocyte-derived macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a poor innate immune response following infection. This study evaluates the effects of transforming growth factor beta 1 (TGFß1) up-regulated by PRRSV on gene expressions of co-stimulatory molecules, type I interferon (IFN), type I IFN-regulated genes (IRGs), pattern recognition receptors, and pro-inflammatory cytokines in PRRSV-inoculated monocyte-derived macrophages (MDMs). Phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) specific to various regions of porcine TGFß1 mRNA were synthesized, and those specific to the AUG region efficiently knockdown TGFß1 mRNA expression and protein translation. Transfection of TGFßAS ODNs in MDMs inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) significantly reduced TGFß1 mRNA expression and significantly increased mRNA expressions of CD80, CD86, IFNß, IRGs (i.e. IFN regulatory factor 3 (IRF3), IRF7, myxovirus resistance 1, osteopontin, and stimulator of IFN genes), Toll-like receptor 3, and tumor necrosis factor-alpha. Transfection of TGFßAS ODNs in MDMs inoculated with HP-PRRSV-2 also significantly increased mRNA expressions of IFNα, IFNγ, and 2'-5'-oligoadenylate synthetase 1. The quantity of PRRSV-2 RNA copy numbers was significantly reduced in MDMs transfected with TGFßAS ODNs as compared to untransfected MDMs. Recombinant porcine TGFß1 (rTGFß1) and recombinant porcine IFNα (rIFNα) sustained and reduced the yields of PRRSV-2 RNA copy numbers in PRRSV-2 inoculated MDMs, respectively. These findings demonstrate a strategy of PRRSV for innate immune suppression via an induction of TGFß expression. These findings also suggest TGFß as a potential parameter that future PRRSV vaccine and vaccine adjuvant candidates should take into consideration.

Type I and II interferons, transcription factors and major histocompatibility complexes were enhanced by knocking down the PRRSV-induced transforming growth factor beta in monocytes co-cultured with peripheral blood lymphocytes.

The innate and adaptive immune responses elicited by porcine reproductive and respiratory syndrome virus (PRRSV) infection are known to be poor. This study investigates the impact of PRRSV-induced transforming growth factor beta 1 (TGFß1) on the expressions of type I and II interferons (IFNs), transcription factors, major histocompatibility complexes (MHC), anti-inflammatory and pro-inflammatory cytokines in PRRSV-infected co-cultures of monocytes and peripheral blood lymphocytes (PBL). Phosphorothioate-modified antisense oligodeoxynucleotide (AS ODN) specific to the AUG region of porcine TGFß1 mRNA was synthesized and successfully knocked down TGFß1 mRNA expression and protein translation. Monocytes transfected with TGFßAS1 ODN, then simultaneously co-cultured with PBL and inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) showed a significant reduction in TGFß1 mRNA expression and a significant increase in the mRNA expressions of IFNα, IFNγ, MHC-I, MHC-II, signal transducer and activator of transcription 1 (STAT1), and STAT2. Additionally, transfection of TGFßAS1 ODN in the monocyte and PBL co-culture inoculated with cPRRSV-2 significantly increased the mRNA expression of interleukin-12p40 (IL-12p40). PRRSV-2 RNA copy numbers were significantly reduced in monocytes and PBL co-culture transfected with TGFßAS1 ODN compared to the untransfected control. The yields of PRRSV-2 RNA copy numbers in PRRSV-2-inoculated monocytes and PBL co-culture were sustained and reduced by porcine TGFß1 (rTGFß1) and recombinant porcine IFNα (rIFNα), respectively. These findings highlight the strategy employed by PRRSV to suppress the innate immune response through the induction of TGFß expression. The inclusion of TGFß as a parameter for future PRRSV vaccine and vaccine adjuvant candidates is recommended.

Oxidative stress related effect of xenobiotics on eukaryotic model organism, Saccharomyces cerevisiae.

Ecotoxicological assays have traditionally focused on the effects of chemicals at the individual level by exploiting mortality and reproduction as endpoints. Although these two parameters are ecologically relevant, they rarely provide information regarding the elemental toxic mechanisms. Obviously, the number of xenobiotics used has been rapidly increased. Thus, any established measurement that shortens the actual outcome and, simultaneously provides information about toxic mechanisms is desirable. This research focused on the study of oxidative stress response as a biomarker in the eukaryotic model organism, Saccharomyces cerevisiae. For this, yeast cells were exposed to a set of selected environmentally relevant chemicals via different approaches, including cellular diagnostics, gene expression analysis and chemo-genetic screening. The results demonstrated that at the cellular level, model organisms reacted to different chemicals in distinct manner. For each xenobiotic, the correlation between toxic response of molecular and cellular levels are presented. Namely, the expression of target genes after chemical exposure affected the cellular alteration as evidenced by an elevated level of superoxide dismutase and a reduced amount of glutathione. Furthermore, the results derived from chemo-genetic screening, in which mutants lacking of gene of interest were employed, exhibited more susceptibility to test chemicals in comparison to the wildtype. The response of oxidative stress upon chemical exposure in budding yeast from this study is potentially useful for an establishment of a proper bio-test system which can eventually be linked to adverse effects at an individual level in higher eukaryotes.

Oral Supplementation of Houttuynia cordata Extract Reduces Viremia in PRRSV-1 Modified-Live Virus-Vaccinated Pigs in Response to the HP-PRRSV-2 Challenge.

This study evaluated the in vitro antiviral activities and the ex vivo immunomodulatory effects of Houttuynia cordata Thunb. (HC) ethanolic extracts in response to porcine reproductive and respiratory syndrome virus (PRRSV). In addition, this study evaluated the in vivo effects of oral supplementation of HC extract on immune responses to and cross-protective efficacy of PRRSV-1 modified-live virus (MLV) vaccine against the highly pathogenic (HP)-PRRSV-2 challenge. In vitro experiments demonstrated that HC extracted in either 50%, 70%, or 95% ethanol (referred to as HC50, HC70, and HC95, respectively) significantly interfered with PRRSV replication in MARC-145 cells. Ex vivo experiments revealed that all HC extracts significantly enhanced mRNA expressions of type I interferon-regulated genes, type I and II interferon (IFN), and pro- and anti-inflammatory cytokines in HP-PRRSV-2-inoculated monocyte-derived macrophages. An in vivo experiment included four groups of six pigs (4 weeks old; n = 24). Group 1 and group 2 were vaccinated with the PRRSV-1 MLV vaccine at 0 dpv (day post vaccination). Group 2 also received oral administration of HC50 extract at 0-49 dpv. Group 3 received the PRRSV-1 MLV vaccine solvent at 0 dpv, while group 4 served as strict control. Groups 1-3 were challenged intranasally with HP-PRRSV-2 at 28 dpv and immune-related and clinical parameters were monitored weekly until 49 dpv. Compared to group 1, group 2 demonstrated significantly increased IFN regulatory factor 3 mRNA expression of PRRSV-recalled peripheral blood mononuclear cells, and significantly reduced HP-PRRSV-2 viremia. No difference in PRRSV-specific antibody responses, rectal temperature, clinical scores, and average daily weight gain was detected. Our study reports the immunomodulatory and anti-PRRSV potentials of HC extract in PRRSV-1 MLV-vaccinated/HP-PRRSV-2 challenged pigs.

Rutin, α-tocopherol, and l-ascorbic acid up-regulate type I interferon-regulated gene and type I and II interferon expressions and reduce inflammatory cytokine expressions in monocyte-derived macrophages infected with highly pathogenic porcine reproductive and respiratory syndrome virus.

This study evaluated the immunomodulatory effect of two types of phytochemicals, i.e. rutin and ß-carotene, and two types of vitamins, i.e. α-tocopherol and l-ascorbic acid on improving innate immune responses to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Monocyte-derived macrophages (MDM) from eight PRRSV-seronegative pigs were inoculated with HP-PRRSV and subsequently stimulated with rutin, ß-carotene, α-tocopherol, and l-ascorbic acid in the absence or presence of either polyinosinic:polycytidylic acid or lipopolysaccharide. The mRNA expression levels of myxovirus resistance 1, interferon regulatory factor 3 (IRF3), IRF7, 2'-5'-oligoadenylatesynthetase 1, stimulator of interferon genes (STING), osteopontin (OPN), interferon alpha (IFNα), IFNß, IFNγ, interleukin-10 (IL-10), tumor necrosis factor alpha (TNFα), and transforming growth factor beta (TGFß) were evaluated by real-time PCR. Compared with control MDM, HP-PRRSV significantly suppressed mRNA expressions of all immune-related genes except IL-10 and TGFß. Compared with HP-PRRSV-inoculated MDM, stimulation with rutin, α-tocopherol, and l-ascorbic acid, but not ß-carotene significantly enhanced mRNA expression levels of IRF3, IRF7, STING, OPN, IFNα, IFNß, and IFNγ in HP-PRRSV-inoculated MDM. Stimulation with rutin also significantly reduced mRNA expression levels of TNFα and TGFß, whereas stimulation with ß-carotene and α-tocopherol significantly reduced TNFα mRNA expression in HP-PRRSV-inoculated MDM. Our findings demonstrate the potentials of rutin, α-tocopherol, and l-ascorbic acid in enhancing type I interferon-regulated genes and type I and II IFN expressions, and in reducing pro- and/or anti-inflammatory cytokine expressions in HP-PRRSV-inoculated MDM. Our findings suggest that rutin, α-tocopherol, and l-ascorbic acid may serve as effective immunomodulators for improving innate immune response to HP-PRRSV.

Adjuvants for swine vaccines: Mechanisms of actions and adjuvant effects.

This review deals with mechanisms of actions (MOA) and adjuvant effects of various types of adjuvants for swine vaccines. A number of different types of adjuvants have been tested with swine vaccines, including oil emulsion, particulate antigen (Ag) carrier, cytokines, pathogen-associated molecular patterns and immune ligands, saponins, and bacterial cells and toxins. In addition, there are a number of chemicals and natural products that possess adjuvant activities when tested with swine vaccines, and are grouped as miscellaneous in this review. The MOA of adjuvants can be generally divided into two categories: delivery vehicles and immunostimulants. Adjuvants serving as delivery vehicle can act as a depot, help deliver Ag to the draining lymph nodes, promote Ag uptake by antigen-presenting cells (APCs), and protect Ag from harsh conditions. Adjuvants possessing immunostimulatory activities may help recruit and activate APCs and T cells, enhance APC functions, and direct T cell differentiation and immunoglobulin isotype switching. Success of adjuvant use on improving immunogenicity and protective efficacy of swine vaccines depends on several factors, including type and stability of vaccine Ag, dose and schedule of vaccination, MOA of adjuvant, route, dose and schedule of adjuvant administration, type of immune response required, and safety from adverse reactions. In addition to the above mentioned factors, cost effectiveness is of concern. Further studies of swine vaccine adjuvants may need to focus on characterization of their MOA and search for more potential adjuvant candidates that can induce mucosal immune response.

Oral supplementation of quercetin in PRRSV-1 modified-live virus vaccinated pigs in response to HP-PRRSV-2 challenge.

This study evaluated the immunomodulatory effect of quercetin on improving cross protection of porcine reproductive and respiratory syndrome virus-1 (PRRSV-1) modified-live virus (MLV) vaccine against highly pathogenic (HP)-PRRSV-2 challenge. Ex vivo experiments demonstrated that quercetin significantly enhanced type I interferon-regulated genes (IRGs) and type I and II interferon (IFN), and significantly decreased pro- and anti-inflammatory cytokine expressions in HP-PRRSV-inoculated monocyte-derived macrophages. In vivo experiments divided pigs (4-week-old; n = 24) into four groups of six pigs. Group 1 and group 2 were immunized with PRRSV-1 MLV vaccine at 0 dpv (day post vaccination). Group 2 also received oral administration of quercetin at 0-49 dpv. Group 3 was injected with PRRSV-1 MLV vaccine solvent at 0 dpv. Group 4 served as strict control. Group 1-3 were challenged intranasally with HP-PRRSV at 28 dpv and immune and clinical parameters were monitored weekly until 49 dpv. Group 1 demonstrated significantly reduced HP-PRRSV viremia, rectal temperature and clinical scores, and significantly improved average daily weight gain (ADWG), compared to group 3. Group 2 demonstrated significantly increased IFN regulatory factor 3, stimulator of IFN genes, IFNα, and significantly decreased transforming growth factor beta (TGFß) mRNA expressions, compared to group 1. The animals demonstrated significantly reduced HP-PRRSV viremia, but did not demonstrate any further improved PRRSV-specific antibody responses, rectal temperature, clinical scores, and ADWG as compared to group 1. Our findings suggest that quercetin up-regulates IRGs, IFNα, and down-regulates TGFß mRNA expressions which may contribute to further reducing number of viremic pigs and HP-PRRSV viremia which were conferred by PRRSV-1 MLV vaccine. Our findings also suggest that quercetin may serve as an effective oral immunomodulator for improving cell-mediated immune defense to HP-PRRSV.

Adjuvants for porcine reproductive and respiratory syndrome virus vaccines.

This review deals with present and past efforts in utilization of vaccine adjuvants for porcine reproductive and respiratory syndrome virus (PRRSV) vaccines. PRRSV vaccines elicit delayed and weak cell-mediated immune (CMI) and antibody responses after vaccination. Several kinds of vaccine adjuvants have been utilized to accelerate and magnify immune responses to PRRSV vaccines. These adjuvants include cytokines, chemical reagents, and bacterial products. Of 11 vaccine adjuvants tested, five (i.e. interleukin-2 (IL-2), IL-12, interferon alpha (IFNalpha), polyinosinic and polycytidylic acid, and cytidine-phosphate-guanosine oligodeoxynucleotides (CpG ODN)) significantly enhance CMI response to PRRSV vaccines. The response is characterized by proliferation, cytotoxicity, and IFNgamma secretion of peripheral blood mononuclear cells or T cells in response to recall PRRSV antigens in vitro. Two (i.e. CpG ODN and cholera toxin) significantly enhance PRRSV-specific antibody response after vaccination. Two (i.e. IL-2 and CpG ODN) significantly enhance protective efficacy of PRRSV vaccines in challenge models. Improvement of immune responses to PRRSV vaccines should focus in future studies on assessing more vaccine adjuvants for their efficiency in enhancing both CMI and antibody responses and on identifying PRRSV components and strategies that down-modulate pig immune responses in order to devise vaccine adjuvants that can regulate such strategies of the virus.

Co-administration of saponin quil A and PRRSV-1 modified-live virus vaccine up-regulates gene expression of type I interferon-regulated gene, type I and II interferon, and inflammatory cytokines and reduces viremia in response to PRRSV-2 challenge.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating virus which suppresses the expression of type I and II interferons (IFNs) as well as several pro-inflammatory cytokines. Our previous study reported that saponin quil A had a potential to up-regulate the expression of type I IFN-regulated genes and type I and II IFNs in porcine peripheral blood mononuclear cells (PBMC) inoculated with PRRSV. The present study evaluated the immunostimulatory effect of quil A on potentiating cross protective immunity of PRRSV-1 modified-live virus (MLV) vaccine against PRRSV-2 challenge. Twenty-four 4-week-old PRRSV-seronegative pigs were divided into four groups of six pigs. Group 1 and group 2 pigs were vaccinated with PRRSV-1 MLV vaccine at 0 dpv (day post vaccination), and additionally group 2 pigs were injected intramuscularly with quil A at -1, 0, 1 dpv. Group 3 pigs were injected with PRRSV-1 MLV vaccine solvent at 0 dpv and served as challenge control, while group 4 pigs served as strict control. Group 1-3 pigs were challenged intranasally with PRRSV-2 at 28 dpv and immune and clinical parameters were observed from 0 until 49 dpv. Group 1 pigs showed significantly reduced PRRSV viremia, number of viremic pigs, and clinical scores, and significantly improved average daily weight gain (ADWG), compared to group 3 pigs. Group 2 pigs showed significantly increased mRNA expressions of interferon regulatory factor 3, 2'-5'-oligoadenylatesynthetase 1, osteopontin, IFNα, IFNß, IFNγ, interleukin-2 (IL-2), IL-13 and tumor necrosis factor alpha, compared to group 1 pigs. The animals demonstrated significantly reduced PRRSV viremia and number of viremic pigs, but did not demonstrate any further improved PRRSV-specific antibody levels, neutralizing antibody titers, rectal temperature, clinical scores, and ADWG as compared to group 1 pigs. Our findings suggest that quil A up-regulates type I IFN-regulated gene, type I and II IFNs, and inflammatory cytokine expressions which may contribute to further reducing PRRSV viremia and number of viremic pigs which were conferred by PRRSV-1 MLV vaccine. Our findings also suggest that quil A may serve as an effective immunostimulator for potentiating cell-mediated immune defense to PRRSV.

Saponin Quil A up-regulates type I interferon-regulated gene and type I and II interferon expressions which are suppressed by porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses innate immune response following infection of myeloid antigen-presenting cells. Poor innate immune response results in weak and delayed PRRSV-specific adaptive immunity, and facilitates PRRSV replication, pathogenesis, and persistent infection. Numerous efforts have been made to enhance the effective innate and adaptive immune defenses to PRRSV, however, only a few attempts have so far elicited satisfactory results. The present study aims to evaluate in vitro the potential of saponin quil A to enhance the expression of type I interferon (IFN)-regulated gene, type I and II IFNs, and pro-inflammatory cytokines in PRRSV-inoculated peripheral blood mononuclear cells (PBMC). Naïve PBMC from four PRRSV-seronegative pigs were inoculated with PRRSV and subsequently stimulated with quil A in the absence or presence of either polyinosinic:polycytidylic acid (poly IC) or lipopolysaccharide (LPS). The mRNA expression levels of myxovirus resistance 1 (Mx1), interferon regulatory factor 3 (IRF3), IRF7, 2'-5'-oligoadenylatesynthetase 1 (OAS1), stimulator of interferon genes (STING), osteopontin (OPN), IFNα, IFNß, IFNγ, interleukin-2 (IL-2), IL-10, IL-13, tumor necrosis factor alpha (TNFα), and transforming growth factor beta (TGFß) were evaluated by real-time PCR. Compared with uninoculated PBMC, PRRSV significantly suppressed expression of all immune parameters except IL-2, IL-10, IL-13, and TGFß. When compared with PRRSV-inoculated PBMC, stimulation with quil A significantly enhanced Mx1, IRF3, IRF7, OAS1, STING, IFNß, and IFNγ mRNA expressions, and significantly reduced TGFß mRNA expression. Our findings thus suggest that quil A has a potential to up-regulate the expression of type I IFN-regulated gene and type I and II IFNs which are suppressed by PRRSV. Therefore, it may serve as an effective immunostimulator for potentiating the innate immune defense to PRRSV.

Effects of porcine reproductive and respiratory syndrome virus-infected antigen-presenting cells on T cell activation and antiviral cytokine production.

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) to suppress T cell expression of CD25 (alpha chain of interleukin [IL]-2 receptor), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) was determined by flow cytometry in naive porcine T cells in response to mitogen (concanavalin A) and cytokine inducers (phorbol 12-myristate 13-acetate plus ionomycin [PMA/I]). Four PRRSV isolates of varying clinical virulence and three different types of porcine myeloid antigen-presenting cells (APCs) were used. T cells cultured with monocytes infected with virulent PRRSV (VR-2385, SDSU-73, and VR-2332), but not with a vaccine strain (Ingelvac PRRS MLV; Boehringer Ingelheim Vetmedica, St. Joseph, MO), demonstrated significantly reduced CD25 expression (%CD25(+)) and IFN-gamma expression (%IFN-gamma (+)) compared with T cells incubated with uninoculated monocyte cultures. T cells cultured with monocytes infected with all four PRRSV isolates demonstrated significantly reduced %TNF-alpha (+). The significant reduction of %CD25(+), %IFN-gamma (+), and %TNF-alpha (+) was not detected in T cells cultured with monocyte-derived macrophages (MDMs) and immature monocyte-derived dendritic cells (MDCs) infected with any PRRSV isolates. Heat-inactivated PRRSV did not induce significantly reduced T cell responses in any APC cultures. The reduction of T cell response in monocyte cultures was not due to PRRSV-induced T cell death. Gene expression of IL-10 detected by semiquantitative reverse transcriptase-polymerase chain reaction was significantly increased in virulent PRRSV-infected monocyte cultures after PMA/I, but not concanavalin A, stimulation compared with IL-10 gene expression from uninoculated monocyte cultures. Increased IL-10 gene expression contributed to significantly reduced %IFN-gamma (+) and %TNF-alpha (+), but not %CD25(+), as determined by IL-10 neutralization assay. This study reports that PRRSV has the ability to suppress T cell responses. The suppressive ability of PRRSV is associated with viral virulence and is mediated by virus-infected monocytes, but not by virus-infected MDMs and immature MDCs.

Immune responses and protection by vaccine and various vaccine adjuvant candidates to virulent porcine reproductive and respiratory syndrome virus.

Various vaccine adjuvant candidates were assessed with the modified-live porcine reproductive and respiratory syndrome virus (MLV PRRSV) (Ingelvac PRRS MLV) vaccine. Their influence on humoral-mediated immune (HMI) and cell-mediated immune (CMI) responses as well as protection from virulent PRRSV challenge (MN-184) was evaluated. Ninety seronegative pigs were randomly divided into nine groups of 10 pigs. One group received MLV vaccine alone. Five groups received MLV vaccine with either bacterial endotoxin-derived adjuvant (ET), mixed open reading frame 5 (ORF5) peptides derived from various PRRSV isolates, porcine interferon alpha (IFNalpha), polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC), or porcine interleukin-12 (IL-12). One group did not receive MLV vaccine but was immunized with ORF5 peptides conjugated with cholera toxin (ORF5 peptide/CT). Two groups served as challenged and unchallenged non-vaccinated controls. Four-color flow cytometry was utilized to simultaneously identify three major porcine T-cell surface markers (CD4, CD8, and gammadelta TCR) and detect activation marker CD25 (alpha chain of IL-2 receptor) or intracellular IFNgamma. The MLV PRRSV vaccine alone successfully primed CD4(-)CD8(+)gammadelta- T-cells as demonstrated by a significant increase in %IFNgamma+ cells when live PRRSV was used as a recall antigen. Booster immunizations of mixed ORF5 peptides and co-administration of IL-12 with MLV PRRSV vaccine significantly enhanced IFNgamma expression by some T-cell subsets (CD4(-)CD8(+)gammadelta+ and CD4(-)CD8(-)gammadelta+ for mixed ORF5 peptides and CD4(+)CD8(+)gammadelta- and CD4(-)CD8(+)gammadelta+ for IL-12). All groups receiving MLV-vaccine with or without adjuvants had reduced lung lesions after challenge. The group immunized with only ORF5 peptide/CT did not have significant T-cell recall responses and was not protected from challenge. Expression of IFNgamma by several T-cell subsets correlated with reduced lung lesions and viremia, whereas expression of CD25 did not. Expression of surface CD25 did not correlate with IFNgamma production. PRRSV ELISA s/p ratio prior to challenge also correlated with reduced lung lesions and viremia. In conclusion, booster immunizations of the mixed ORF5 peptides and co-administration of IL-12 effectively enhanced the CMI response to MLV vaccine. However, neither adjuvant significantly contributed to reducing clinical effects when compared to MLV alone.

Biology of porcine T lymphocytes.

The present review concentrates on the biological aspects of porcine T lymphocytes. Their ontogeny, subpopulations, localization and trafficking, and responses to pathogens are reviewed. The development of porcine T cells begins in the liver during the first trimester of fetal life and continues in the thymus from the second trimester until after birth. Porcine T cells are divided into two lineages, based on their possession of the alphabeta or gammadelta T-cell receptor. Porcine alphabeta T cells recognize antigens in a major histocompatibility complex (MHC)-restricted manner, whereas the gammadelta T cells recognize antigens in a MHC non-restricted fashion. The CD4+CD8- and CD4+CD8lo T cell subsets of alphabeta T cells recognize antigens presented in MHC class II molecules, while the CD4-CD8+ T cell subset recognizes antigens presented in MHC class I molecules. Porcine alphabeta T cells localize mainly in lymphoid tissues, whereas gammadelta T cells predominate in the blood and intestinal epithelium of pigs. Porcine CD8+ alphabeta T cells are a prominent T-cell subset during antiviral responses, while porcine CD4+ alphabeta T cell responses predominantly occur in bacterial and parasitic infections. Porcine gammadelta T cell responses have been reported in only a few infections. Porcine T cell responses are suppressed by some viruses and bacteria. The mechanisms of T cell suppression are not entirely known but reportedly include the killing of T cells, the inhibition of T cell activation and proliferation, the inhibition of antiviral cytokine production, and the induction of immunosuppressive cytokines.

Effects of dietary Centella asiatica (L.) Urban on growth performance, nutrient digestibility, blood composition in piglets vaccinated with Mycoplasma hyopneumoniae.

To investigate the effects of Centella asiatica (L.) on growth performance, nutrient digestibility and blood composition in piglets, 32 nursery pigs were fed 0.0, 0.5, 1.0 and 2.0% dietary C. asiatica (L.) from 15 to 90 kg BW. At 30 kg BW, nutrient digestibility was measured and at 35 kg BW piglets were vaccinated with Mycoplasma hyopneumoniae. Hematological parameters were checked at 40 and 80 kg BW. Compared with the control, growth performance was not affected. The ether extract, ash and calcium digestibility were lower at 0.5%, and dry matter, crude protein, crude fat, phosphorus and energy digestibility were lower at 1.0% (P<0.05). On hematological values, at 40 kg hematocrit, total white blood cells, neutrophils, eosinophils, basophils, monocytes and lymphocytes were higher at the 2.0% level (P<0.05). Most of these values except basophils and monocytes continued until at 80 kg, at which total white blood cells, neutrophils, eosinophils and lymphocytes were higher even at 1.0% (P<0.05); neutrophil-to-lymphocyte ratio tended to be higher at 2.0% (P<0.03). Cholesterol, triglycerides and antibody levels against M. hyopneumoniae did not differ except that at 40 kg the cholesterol of 0.5% was lower (P<0.05) and M. hyopneumoniae-specific antibodies tended to be higher with increasing levels of C. asiatica (L.) (P<0.07). The result that C. asiatica (L.) could not improve growth performance but increased values of serum hematocrit and white blood cells, and mycoplasma immunity to M. hyopneumoniae might suggest that C. asiatica (L.) has no function to elevate body weight but has the potential to enhance innate immunity.

PCV2 on the spot-A new method for the detection of single porcine circovirus type 2 secreting cells.

A porcine circovirus type 2 SPOT (PCV2-SPOT) assay was established to enumerate virus-secreting lymphocytes obtained from naturally infected pigs. The assay is based on the same principle as general ELISPOT assays but instead of detecting cytokine or immunoglobulin secretion, PCV2 particles are immobilized and detected as filter spots. The method was used to evaluate the influence of various cell activators on the PCV2 secretion in vitro and was also applied to study the PCV2 secretion by lymphocytes obtained from pigs in healthy herds and in a herd afflicted by postweaning multisystemic wasting disease (PMWS). Peripheral blood mononuclear cells (PBMCs) obtained from a pig with severe PMWS produced PCV2-SPOTs spontaneously whereas PBMCs obtained from pigs infected subclinically only generated PCV2-SPOTs upon in vitro stimulation. The PCV2 secretion potential was related to the PCV2 DNA content in the PBMCs as determined by two PCV2 real-time PCR assays, developed to differentiate between Swedish PCV2 genogroups 1 (PCV2a) and 3 (PCV2b). Besides the current application these qPCRs could simplify future epidemiological studies and allow genogroup detection/quantitation in dual infection experiments and similar studies. The developed PCV2-SPOT assay offers a semi-quantitative approach to evaluate the potential of PCV2-infected porcine cells to release PCV2 viral particles as well as a system to evaluate the ability of different cell types or compounds to affect PCV2 replication and secretion.

Early inflammatory response to the saponin adjuvant Matrix-M in the pig.

The early inflammatory response to Matrix-M was evaluated in pigs. Adverse reactions measured as body temperature, appetite, activity level and reaction at the site of injection were not observed after s.c. injection with three doses of the adjuvant (75, 100 or 150µg) into one week old piglets. Analyses of the immediate cytokine response of PBMC after in vitro exposure to Matrix-M (AbISCO-100(®)) revealed only a low expression of mRNA for tumour necrosis factor-α (p<0.05) after 6h incubation. Histological examination revealed an infiltration of leukocytes, haemorrhage and necrosis in muscle 24h after i.m. injection of 150µg Matrix-M in pigs aged eleven weeks. At this time, different grades of reactive lymphoid hyperplasia were recorded in the draining lymph node that was enlarged in three of these six pigs injected with Matrix-M. The global transcriptional response at the site of injection and in the draining lymph node was analyzed using Affymetrix GeneChip Porcine Genome Array. A significant enrichment of gene signatures for the cell types described as "myeloid cells" and "plasmacytoid dendritic cells" was observed at the site of injection in Matrix-M injected pigs compared with pigs injected with saline. A number of genes encoding cytokines/chemokines or their receptors were upregulated at the injection site as well as in the draining lymph node. In the draining lymph node, a majority of the upregulated genes were interferon-regulated genes (IRGs). The expression of IFN-ß, but not IFN-α, was increased in the draining lymph nodes of a majority of the pigs exposed to Matrix-M. These IFN-ß expressing pigs also expressed increased levels of osteopontin (OPN) or stimulator of interferon genes (STING), two factors known to facilitate the expression of type I IFNs in response to viral infection. Thus, Matrix-M does not appear to induce any harmful inflammatory response in piglets whilst contributing to the innate immunity by activating the type I IFN system, possibly through several alternative signalling pathways.

Plasmids expressing porcine interferon gamma up-regulate pro-inflammatory cytokine and co-stimulatory molecule expression which are suppressed by porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses the pro-inflammatory immune response following infection of myeloid antigen-presenting cells. A reduced pro-inflammatory immune response modulates PRRSV replication, clinical disease, and persistent infection of the virus. Numerous efforts have been made to enhance the pro-inflammatory immune response to PRRSV, but only a few attempts have so far elicited satisfactory results. The present study aims to evaluate in vitro the potential of plasmids expressing porcine interferon gamma (pcDNA-IFNγ) to enhance the expression of pro-inflammatory immune parameters in PRRSV-inoculated monocytes. Naïve blood monocytes from eight PRRSV-seronegative pigs were inoculated with PRRSV and subsequently transfected with pcDNA-IFNγ or pcDNA (empty plasmid vector) and stimulated with lipopolysaccharide (LPS). The mRNA expression levels of IFNγ, interleukin-1 beta (IL-1ß), IL-10, IL-12p40, tumor necrosis factor alpha (TNFα), transforming growth factor beta (TGFß), CD80, and CD86 were evaluated by real-time PCR. The IFNγ, IL-10, and TNFα protein production was determined by ELISA. Compared with PRRSV-inoculated monocyte control, transfection with pcDNA-IFNγ, but not pcDNA, significantly enhanced IFNγ, TNFα, CD80, and CD86 mRNA expression, and IFNγ and TNFα protein production. A slight increase in IL-1ß and IL-12p40 mRNA expression was also observed. Neither pcDNA-IFNγ nor pcDNA transfection affected IL-10 and TGFß expression. Our results thus suggest that pcDNA-IFNγ may be an effective immunostimulator for potentiating the pro-inflammatory immune response to PRRSV.

Porcine reproductive and respiratory syndrome virus vaccines: Immunogenicity, efficacy and safety aspects.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is the leading cause of economic casualty in swine industry worldwide. The virus can cause reproductive failure, respiratory disease, and growth retardation in the pigs. This review deals with current status of commercial PRRS vaccines presently used to control PRRS. The review focuses on the immunogenicity, protective efficacy and safety aspects of the vaccines. Commercial PRRS modified-live virus (MLV) vaccine elicits delayed humoral and cell-mediated immune responses following vaccination. The vaccine confers late but effective protection against genetically homologous PRRSV, and partial protection against genetically heterologous virus. The MLV vaccine is of concern for its safety as the vaccine virus can revert to virulence and cause diseases. PRRS killed virus (KV) vaccine, on the other hand, is safe but confers limited protection against either homologous or heterologous virus. The KV vaccine yet helps reduce disease severity when administered to the PRRSV-infected pigs. Although efforts have been made to improve the immunogenicity, efficacy and safety of PRRS vaccines, a better vaccine is still needed in order to protect against PRRSV.

Plasmids expressing interleukin-10 short hairpin RNA mediate IL-10 knockdown and enhance tumor necrosis factor alpha and interferon gamma expressions in response to porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been suggested to exploit interleukin-10 (IL-10) to suppress immune defense of infected pigs. The present study constructed plasmids encoding selected short hairpin RNA specific to porcine IL-10 mRNA (pIL-10sh) to knockdown IL-10 transcription and investigated the suppressive effect of PRRSV-induced IL-10 on various immune marker expressions. Naïve blood monocytes from eight PRRSV-seronegative pigs were transfected with pIL-10sh and pNeg (plasmid vector) prior to PRRSV inoculation and subsequent lipopolysaccharide (LPS) stimulation. The mRNA expressions of IL-10, IL-1ß, IL-12p40, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), transforming growth factor beta (TGFß), CD80, and CD86 were evaluated by real-time PCR. The IL-10, TNFα, and IFNγ protein productions were determined by ELISA. Compared with non-transfected monocyte control, transfection with selected pIL-10sh (pIL-10sh1), but not other pIL-10sh nor pNeg, significantly reduced IL-10 expression and significantly enhanced TNFα and IFNγ expressions. Slight increases in IL-1ß, IL-12p40, CD80, and CD86 expressions were also observed. Neither pIL-10sh1 nor pNeg transfection affected TGFß expression. Our results indicate that PRRSV does exploit IL-10 to suppress the expressions of pro-inflammatory cytokines, mainly TNFα and IFNγ, and co-stimulatory molecules, CD80 and CD86.

Interleukin-10 antisense oligodeoxynucleotide suppresses IL-10 expression and effects on proinflammatory cytokine responses to porcine reproductive and respiratory syndrome virus.

Upregulation of interleukin-10 (IL-10) expression has been suggested to be the mechanism by which the porcine reproductive and respiratory syndrome virus (PRRSV) suppresses the innate and adaptive immune response in infected pigs. In this study we evaluated the potential of phosphorothioate-modified IL-10 antisense oligodeoxynucleotide specific to the translation initiation region of porcine IL-10 mRNA (IL-10AS) in enhancing proinflammatory cytokine responses to PRRSV. Naïve peripheral blood mononuclear cells from eight PRRSV-seronegative pigs were transfected with IL-10AS in vitro prior to PRRSV inoculation and phorbol 12-myristate 13-acetate plus ionomycin or concanavalin A stimulation. The effects of IL-10AS on mRNA expression of IL-10, interferon-gamma (IFN-gamma), IFN-alpha, tumor necrosis factor-alpha (TNF-alpha), IL-2, and IL-4 were tested by real-time PCR. The percentages of IFN-gamma-producing T-cell subsets were determined by flow cytometry. Compared to the controls, the levels of IL-10 and IL-2 mRNA were significantly reduced, while those of IFN-gamma mRNA were increased, and TNF-alpha, IFN-alpha, and IL-4 mRNA were unchanged. An increase in the percentage of the IFN-gamma+ population was also observed in lymphocytes and CD8beta+ T cells. Our results suggest that IL-10AS has the potential to enhance proinflammatory cytokine responses to PRRSV infection.