Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3⁺CD8^high cells starting from 14 dpi. Although CD3⁺CD8^high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3⁺CD8^high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.     

The combination of PRRS virus and bacterial endotoxin as a model for multifactorial respiratory disease in pigs

This paper reviews in vivo studies on the interaction between porcine reproductive and respiratory syndrome virus (PRRSV) and LPS performed in the authors' laboratory. The main aim was to develop a reproducible model to study the pathogenesis of PRRSV-induced multifactorial respiratory disease. The central hypothesis was that respiratory disease results from an overproduction of proinflammatory cytokines in the lungs. In a first series of studies, PRRSV was shown to be a poor inducer of TNF-alpha and IFN-alpha in the lungs, whereas IL-1 and the anti-inflammatory cytokine IL-10 were produced consistently during infection. We then set up a dual inoculation model in which pigs were inoculated intratracheally with PRRSV and 3-14 days later with LPS. PRRSV-infected pigs developed acute respiratory signs for 12-24h upon intratracheal LPS inoculation, in contrast to pigs inoculated with PRRSV or LPS only. Moreover, peak TNF-alpha, IL-1 and IL-6 titers were 10-100 times higher in PRRSV-LPS inoculated pigs than in the singly inoculated pigs and the cytokine overproduction was associated with disease. To further prove the role of proinflammatory cytokines, we studied the effect of pentoxifylline, a known inhibitor of TNF-alpha and IL-1, on PRRSV-LPS induced cytokine production and disease. The clinical effects of two non-steroidal anti-inflammatory drugs (NSAIDs), meloxicam and flunixin meglumine, were also examined. Pentoxifylline, but not the NSAIDs, significantly reduced fever and respiratory signs from 2 to 6h after LPS. The levels of TNF-alpha and IL-1 in the lungs of pentoxifylline-treated pigs were moderately reduced, but were still 26 and 3.5-fold higher than in pigs inoculated with PRRSV or LPS only. This indicates that pathways other than inhibition of cytokine production contributed to the clinical improvement. Finally, we studied a mechanism by which PRRSV may sensitize the lungs for LPS. We hypothesized that PRRSV would increase the amount of LPS receptor complex in the lungs leading to LPS sensitisation. Both CD14 and LPS-binding protein, two components of this complex, increased significantly during infection and the amount of CD14 in particular was correlated with LPS sensitisation. The increase of CD14 was mainly due to infiltration of strongly CD14-positive monocytes in the lungs. The PRRSV-LPS combination proved to be a simple and reproducible experimental model for multifactorial respiratory disease in pigs. To what extent the interaction between PRRSV and LPS contributes to the development of complex respiratory disease is still a matter of debate.     

Interleukin-12 (IL-12) ameliorates the effects of porcine respiratory and reproductive syndrome virus (PRRSV) infection

Porcine respiratory and reproductive syndrome virus (PRRSV) disease, one of the most economically significant viral diseases in the swine industry, is characterized by miscarriages, premature farrowing, stillborn pigs, and respiratory disease associated with death and chronic poor performance of nursing and weaned pigs. Interleukin-12 (IL-12) is a key component in driving the development of cell-mediated immunity as well as stimulating interferon-gamma (IFN-gamma) production from T cells and natural killer cells. Although some studies have investigated the use of IL-12 as a vaccine adjuvant in swine, little is known about its effectiveness as a treatment against viral diseases in swine. The present study investigated whether recombinant porcine IL-12 (rpIL-12) enhances the immune response and thereby diminishes the effects of PRRSV infection in young pigs. Interestingly, in vitro experiments demonstrated that rpIL-12 is capable of inducing swine pulmonary alveolar macrophages (PAMs), the target cells of PRRSV, to produce IFN-gamma in a dose and time dependent manner. In addition, in vitro studies also revealed that rpIL-12 treatment was capable of significantly reducing PRRSV viral titers in PAMs. In vivo administration of rpIL-12 significantly decreased PRRSV titers in the lungs and blood of infected animals. Furthermore, treatment with rpIL-12 prevented significant growth retardation in PRRSV-infected animals. Finally, in response to viral antigen recall challenge, PAMs isolated from rpIL-12-treated/PRRSV-infected animals produced greater amounts of IFN-gamma and lesser amounts of interleukin-10 than PAMs isolated from non-rpIL-12-treated/PRRSV-infected animals. Taken together our data indicate that treatment with rpIL-12 may provide an effective approach to control or ameliorate PRRSV-induced disease in swine.     

Use of ELISPOT and ELISA to evaluate IFN-gamma, IL-10 and IL-4 responses in conventional pigs

ELISPOT and ELISA were standardised for pig interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and interleukin-4 (IL-4) with the aim to study the evolution of the immune response in conventional pigs from birth to 6 months of age and also to compare results of both techniques. Five pigs were bled at 1, 6, 9, 12 and 22 weeks of age and peripheral blood mononuclear cells (PBMC) were stimulated with phytohemagglutinin. The frequencies of cytokine secreting cells (CSC) and the levels of secreted cytokines were compared. For IFN-gamma the mean of CSC increased with age (p<0.05) from an average of 486/10(6) PBMC at first week of age to 1256/10(6) PBMC at 22 weeks of age. No correlation was found between the number of IFN-gamma CSC and the cytokine levels obtained by ELISA. For IL-10, frequencies of CSC did not increase with age of pigs, having a low of 315/10(6) PBMC at first week of age and a high of 1485/10(6) PBMC at six weeks. Comparison of ELISA and ELISPOT results for IL-10 showed a certain degree of correlation (r=0.74; p<0.05). Spontaneous secretion was observed in unstimulated cultures. For IL-4, frequencies of CSC were low (50-70/10(6) PBMC). In this case, comparison of ELISA and ELISPOT could not be done because cytokine levels in culture supernatants were often below the detection limit of the IL-4 ELISA. All these values can serve as a reference for future studies and also, our observations suggest that ELISPOT and ELISA should be carefully interpreted and do not necessarily correlate.     

Mannan oligosaccharide increases serum concentrations of antibodies and inflammatory mediators in weanling pigs experimentally infected with porcine reproductive and respiratory syndrome virus

Mannan-containing products are capable of modulating immune responses in animals. However, different products may have diverse immunomodulation. The experiment was conducted to examine effects of mannan oligosaccharide (Actigen; ACT) on growth performance and serum concentrations of antibodies and inflammatory mediators in weanling pigs (Sus scrofa) experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV). A total of 32 PRRSV-negative pigs (3 wk old) were randomly assigned from within blocks to 1 of 4 treatments in a 2 by 2 factorial arrangement [2 types of diet: control (0%) and ACT addition (0.04%); and with and without PRRSV] in a randomized complete block design. Pigs were blocked by initial BW within sex. Ancestry was equalized across treatments. Pigs (8/treatment) were kept individually in each pen. After 2 wk of an 8-wk period of feeding the treatments, pigs received an intranasal inoculation of PRRSV or sham medium at 5 wk of age. Infection by PRRSV decreased ADG, ADFI, and G:F throughout the experiment (P < 0.01). Actigen did not affect ADG (P = 0.450), but decreased (P = 0.047) ADFI from 28 to 42 days postinoculation (DPI). During that time, ACT improved G:F in infected pigs but not in sham controls (interaction, P = 0.009). Dietary ACT did not affect viremia in infected pigs (P > 0.05), but increased PRRSV-specific antibody titer at 35 DPI (P = 0.042). Infection with PRRSV induced the febrile responses of pigs from 3 to 10 DPI (P < 0.001) with return to normal at 14 DPI. During the experimental period, the rectal temperature of pigs was found slightly elevated by ACT (P = 0.045). Infected pigs had greater serum concentrations of IL-1β, tumor necrosis factor (TNF)-α, IL-12, interferon (IFN)-γ, IL-10, and haptoglobin (Hp) than sham controls (P < 0.001). These results indicate that PRRSV stimulated secretion of cytokines involved in innate, T-helper 1, and T-regulatory immune responses. Actigen tended to decrease the serum TNF-α concentration regardless of PRRSV (P = 0.058). The ACT × PRRSV interaction was significant for IL-1β (P = 0.016), IL-12 (P = 0.026), and Hp (P = 0.047), suggesting that infected pigs fed ACT had greater serum concentrations of these mediators than those fed the control. The increases in IL-1β and IL-12 may favorably promote innate and T-cell immune functions in infected pigs fed ACT. Feeding ACT may be useful as ACT is related to increased PRRSV antibody titers and G:F in infected pigs at certain times during infection.     

Mannan oligosaccharide improves immune responses and growth efficiency of nursery pigs experimentally infected with porcine reproductive and respiratory syndrome virus

This study was conducted to determine whether the ingestion of mannan oligosaccharide (MOS, Bio-Mos) alters the immune response of nursery pigs challenged with porcine reproductive and respiratory syndrome virus (PRRSV). A total of 64 pigs (3 wk old), free of PRRSV, were used in 2 separate but similar experiments conducted sequentially. Pigs were blocked by initial BW. Sex and ancestry were equalized across treatments. Pigs were randomly assigned from within blocks to 1 of 4 treatments in a 2 × 2 factorial arrangement [2 types of diet: control (0%) and MOS addition (0.2%); 2 levels of PRRSV: with and without]. There were 8 replicate chambers of 2 pigs each. After 2 wk of a 4-wk period of feeding the treatments, pigs were intranasally inoculated with PRRSV or a sterile medium at 5 wk of age. The PRRSV challenge decreased ADG, ADFI, and G:F throughout the experiment (P < 0.001). Feeding MOS improved G:F of the pigs during d 7 to 14 (P=0.041) postinfection (PI). Serum concentrations of tumor necrosis factor (TNF)-α, C-reactive protein, and haptoglobin were increased by PRRSV (P < 0.001). The MOS × PRRSV interaction was significant for TNF-α at d 14 PI (P=0.028), suggesting that infected pigs fed MOS had less TNF-α than those fed the control. Dietary MOS increased serum IL-10 at d 14 PI (P=0.036). Further, MOS-fed pigs had greater numbers of white blood cells (WBC) at d 3 (P=0.048) and 7 PI (P=0.042) and lymphocytes at d 7 PI (P=0.023) than control-fed pigs. In contrast, PRRSV decreased (P < 0.01) WBC numbers until d 14 PI. Dietary MOS appeared (P=0.060) to increase the neutrophils in PRRSV-infected pigs at d 3 PI, but no (P=0.202) MOS × PRRSV interaction was found. Infection with PRRSV increased rectal temperature (RT) of pigs at d 3 PI (P < 0.001) and continued to affect the infected pigs fed the control diet until d 14 PI. The MOS × PRRSV interaction for RT was found at d 7 (P < 0.01) and 10 (P=0.098) PI, indicating that the infected pigs fed MOS had a decreased RT compared with those fed the control. This could explain why feed efficiency was improved by MOS. No effect (P > 0.05) of treatments on viremia or PRRSV-specific antibody was observed. These results suggest that MOS is associated with rapidly increased numbers of WBC at the early stage of infection and alleviates PRRSV-induced effects on G:F and fever. The results also indicate that the reduced intensity of inflammation by MOS may be related to changes in inflammatory mediator levels at the end of the acute phase.     

Differential production of proinflammatory cytokines: in vitro PRRSV and Mycoplasma hyopneumoniae co-infection model

An in vitro culture system was developed to investigate the induction of proinflammatory cytokines by Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV). M. hyopneumoniae infected porcine tracheal ring explants were co-cultured with PRRSV infected pulmonary alveolar macrophages (PAMs) for 24h to assess the cytokine production of each pathogen alone and the interaction between the two pathogens in vitro. Semiquantitative RT-PCR was used to measure interleukin (IL) 1alpha, IL1beta, IL6, IL8, IL10, IL12 and tumor necrosis factor (TNF) alpha mRNA in PAMs. Commercial ELISAs were used to measure soluble IL1beta, IL8, IL10 and TNF in the culture supernatant. In the dual infected group, mRNA expression of IL1alpha, IL1beta, IL8 and TNF was increased. Both the M. hyopneumoniae- and PRRSV-infected only groups tended to have increased expression of IL1alpha, IL1beta and IL8 mRNA, although no statistical difference was observed. Increased levels of IL1beta, IL8 and IL10 were present in the supernatant of the dual infected group as measured by ELISA. No increase in soluble TNF was observed in any of the groups. IL8 levels appeared high in all groups independent of infection status. The cause of the elevated IL8 was unknown, however, it may have been a non-specific response by the cells to tissue damage during the harvesting of the tracheal rings. Correlation between mRNA expression and the soluble cytokine levels were similar in the dual infected groups with the exception of IL10 and TNF. Levels of mRNA and soluble protein levels in the single pathogen infected groups were not as consistent. The increased production of proinflammatory cytokines IL1alpha, IL1beta, IL8 and TNF in the group infected with both M. hyopneumoniae and PRRSV suggests that cytokine induced inflammation may play an important role in the severe, chronic pneumonia induced by the concurrent infection of the two pathogens.     

Lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (PRRSV) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (PBMC) from pigs vaccinated and exposed to natural infection

The present study evaluated the lymphocyte activation in PRRSV-vaccinated pigs subsequently exposed to natural infection by in vitro stimulation of peripheral blood mononuclear cells (PBMC) with homologous vaccine and two heterologous PRRSV isolates. The responsiveness was assessed by determining IFN-γ secreting cells by ELISpot assay, lymphocyte CD8 phenotype by intracellular staining/flow cytometry, cytokine gene expression by real-time quantitative PCR and cytokine secretion by ELISA. Conventional pigs were weaned at 28 days of age and inoculated intramuscularly (IM) or needle-less intradermally (ID) with a modified-live PRRSV vaccine suspended in adjuvant, while control pigs were injected with adjuvant alone (ADJ). Blood samples were collected at vaccination, 35 days post-vaccination and after 35 days post-exposure to natural infection by a heterologous field strain. Thirty-five days post-vaccination, PRRSV vaccine induced a low but significant virus-specific IFN-γ secreting cell response upon stimulation with both the vaccine strain and the two isolates in vaccinated pigs. Conversely, after 35 days post-exposure, only the vaccine strain and the BS/114/S isolate triggered this response. Intracellular staining showed that PRRSV-specific immune cells reacting upon vaccine strain and BS/114/S stimulation were mostly CD8⁺ IFN-γ producing cells whereas the stimulation with BS/55 isolate induced an IFN-γ production associated to the CD8^?IFN-γ⁺ phenotype. At 35 days post-vaccination, PBMC from vaccinated pigs showed lower IL-10 expression and release, and higher TNF-α gene expression upon stimulation with both the vaccine and viral isolates. After infection, both cytokines were not differently modulated in different groups. Immune parameters give evidence that IFN-γ secreting cells in the peripheral blood can be elicited upon PRRSV infection although vaccination itself does not stimulate high levels of these reactive cells. Moreover, the cross-reactivity against divergent PRRS viruses can show a different intensity and be differently associated with cytotoxic CD8⁺IFN-γ⁺ as well as CD8^?IFN-γ⁺ cells. Overall, the obtained data confirmed that the immune activation against PRRSV is not dependent on the genetic divergence of the virus. Especially after infection, a different immune reactivity was evident upon stimulation with the different isolates in terms of frequency and CD8 phenotype of PRRSV-specific IFN-γ producing cells. The modulation of cytokines in vaccinated pigs appeared to be more dependent on vaccination or infection conditions than on stimulation by different isolates, and the changes of IL-10 more relevant than those of TNF-α at gene and protein levels. Moreover, under the conditions of this study, the PRRSV vaccine administered via the intradermal route by a needle-less device was confirmed to induce an immune response comparable or in some cases higher than the intramuscular route.     

Systemic cytokine profile in feeder pigs suffering from natural postweaning multisystemic wasting syndrome (PMWS) as determined by semiquantitative RT-PCR and flow cytometric intracellular cytokine detection

Postweaning multisystemic wasting syndrome (PMWS) is an economically important disease in pigs caused by porcine circovirus type 2 (PCV2). Development of this disease is presumably associated with an impairment of the immune system. We, therefore, investigated the systemic expression of relevant cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, TNF-alpha, IFN-gamma) and IL-2Ralpha at mRNA (semiquantitative RT-PCR) and at protein level (flow cytometric intracellular cytokine detection after short-time stimulation of peripheral blood mononuclear cells) in 10 feeder pigs aged 14 weeks suffering from natural PMWS and in 10 clinically healthy pen-mates. Hematological examination revealed a significant (p < 0.001) relative lymphopenia in the diseased animals when compared to reference pigs. IL-1alpha and IL-10 mRNA levels were notably increased in the affected pigs, whereas IL-2 and IL-2Ralpha (CD25) mRNA levels tended to be down-regulated. IL-8, TNF-alpha and IFN-gamma mRNA expressions appeared to be slightly increased. Intracellular cytokine levels as measured by flow cytometry revealed an increase of IL-1beta, IL-2, and IL-6, whereas IL-12 and TNF-alpha expressions were not affected. IFN-gamma was slightly decreased in the diseased animals. In conclusion, despite the assumption, that the cellular immune response to PMWS as a virus-induced disease should be characterized by either a Th1 driven cytokine profile or a cytokine profile indicative of T cell immunosuppression, our results did not support that hypothesis. Nevertheless, data from intracellular cytokine detection suggest an even increased percentage of the remaining lymphocytes capable to produce IL-2 upon in vitro stimulation, which is in contrast to the slightly diminished IL-2 mRNA levels reflecting the in vivo situation at least at the mRNA level.     

Commercial spray-dried porcine plasma does not transmit porcine circovirus type 2 in weaned pigs challenged with porcine reproductive and respiratory syndrome virus

The objective of this study was to evaluate if spray dried porcine plasma (SDPP) containing porcine circovirus type 2 (PCV2) genome supplemented in feed could transmit PCV2 to pigs challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Twenty-three PRRSV-free pigs, non-viraemic for PCV2, were housed in bio-safety level 3 facilities and assigned to four groups in a 2×2 factorial design consisting of PRRSV challenge and a negative control. The diet contained 0 or 8kg SDPP per 100kg of feed. PRRSV challenge groups were inoculated intranasally with 2mL of a suspension containing 10(6) TCID(50)/mL PRRSV. The SDPP used in the study contained 7.56×10(5) PCV2 genome copies per gram. Dietary treatments were fed from 4days prior to PRRSV inoculation until 28days post-inoculation (PI). All challenged pigs developed PRRSV viraemia by day 3PI and PRRSV antibodies were detected in sera by day 14PI, with no difference between diet treatments. Neither PRRSV viraemia nor seroconversion was observed in non-challenged pigs. PCV2 was not detected in the serum of any pigs throughout the experimental period. SDPP containing the PCV2 genome supplemented in feed did not result in PCV2 transmission to either healthy or PRRSV-infected pigs under these experimental conditions.     

Vitamin E supplementation does not mitigate the acute morbidity effects of porcine reproductive and respiratory syndrome virus in nursery pigs

The objective of this study was to determine whether feeding a vitamin E-rich diet would benefit nursery pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). Sixty-four pigs were subjected to one of four treatment combinations (2 x 2 factorial) of dietary vitamin E (adequate or excess) and PRRSV (medium or inoculation with VR-2385 isolate P-129). Pigs were fed experimental diets during a 3-wk period before inoculation as well as during a 12-d period after inoculation. Growth performance was determined throughout the study, and lipid peroxidation in liver, glutathione peroxidase (GPX) activity in serum, circulating white blood cells, and serum interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) were determined in samples collected from pigs killed 4 or 12 d after inoculation. Infection by PRRSV (P < 0.001) induced a marked decrease in both ADFI and ADG, but neither the main effect of diet nor the diet x PRRSV interaction was significant. Neither diet nor PRRSV affected feed efficiency. At 12 d after inoculation, lipid peroxidation in liver and GPX activity in serum were lower in pigs fed excess vitamin E than in those fed adequate vitamin E (P < 0.01), suggesting that the diet high in vitamin E bolstered the antioxidant status of the pigs. However, PRRSV did not affect lipid peroxidation in liver or serum GPX activity, and the diet x PRRSV interaction was not significant. White blood cell counts were decreased and IFN-gamma, and IL-1beta were increased (P < 0.05) 4 and 12 d after inoculation in PRRSV-infected pigs, but neither diet nor the diet x PRRSV interaction was significant. Collectively, these results indicate that increasing antioxidant defenses by feeding high levels of vitamin E did not ameliorate the effects of PRRSV on decreased growth, leukopenia, and increased serum IL-1beta and IFN-gamma. Thus, feeding nursery pigs a diet high in vitamin E may not be useful for mitigating the acute morbidity effects of PRRSV infection.     

One year's study of dynamic and evolution of types I and II PRRSV in a swine farm

This study was to investigate dynamic and evolution of PRRSV in a seed-stock farm by monitoring PRRSV status from 11 June 2009 to 4 August 2010. For laboratory test, around 18-24 umbilical cords from farrowed sows and 5-95 sera from nursery and grow/finish pigs were submitted around every 2 weeks interval during the study. The submitted samples were tested for PRRSV using IDEXX PRRS 2XR ELISA kit, RT-nested PCR. The PRRSV-positive samples were further sequences based on ORF5 and analyzed using MEGA 3.1 program and Beast 1.5.4 package. The surveyed farm was first infected with type II PRRSV but it was infected newly with type I PRRSV of unknown origin, showing rapid substitution to type I PRRSV as a dominant strain in 2 weeks. The type I PRRSV was first detected from umbilical cord of a farrowed sow in 12 January 2010, and secondly from nursery pigs in 26 January 2010. Although sudden increase of mean S/P ratio was found in grow/finish pigs around 2 months earlier than first type I PRRSV detection, no type I PRRSV viremia was found. Thirty three ORF5 full sequences from 14 type II to 19 type I PRRSVs were obtained chronologically in this farm and the genetic characteristics and evolution rates of those sequences were analyzed. The substitution rates (/site/day) of two types were 4.03×10(-5) (type I), 3.09×10(-5) (type II), respectively, which was more frequent than previous reports. The calculated divergence time of type I PRRSV was consistent with the time when the sudden elevation of serum IgG in grow/finish barn was first observed. This study provided fundamental data for type I PRRSV dynamic in a previously type II PRRSV-infected farm and suggested grow/finisher barn could be a primary site for PRRSV introduction.     

Cytokine expression, glucocorticoid and growth hormone changes after porcine reproductive and respiratory syndrome virus (PRRSV-1) infection in vaccinated and unvaccinated naturally exposed pigs

The objective of this paper was to study the changes of some cytokines and neuroendocrine hormones in vaccinated and unvaccinated pigs that were naturally infected by a PRRSV-1 (porcine reproductive and respiratory syndrome virus) heterologous field strain. We analyzed gene expression of pro-inflammatory (TNF-α, IL-1β, MCP-1, IL-6), pro-immune (IFN-γ) and anti-inflammatory cytokines (IL-10) in PBMC, as well as hormonal (GH and cortisol) levels in blood samples of pigs obtained in a field trial previously reported [Martelli P, Gozio S, Ferrari L, Rosina S, De Angelis E, Quintavalla C, et al. Efficacy of a modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in pigs naturally exposed to a heterologous European (Italian cluster) field strain: clinical protection and cell-mediated immunity. Vaccine 2009;27:3788-99]. All vaccinated pigs showed an increase in pro-inflammatory and pro-immune cytokine gene expression with respect to controls and a prompt increase in GH that could be consistently associated with pro-inflammatory cytokines in sustaining innate immunity; moreover, the higher levels of cortisol indicates the activation of the hypothalamus-pituitary-adrenal (HPA) axis response. In contrast, unvaccinated pigs showed down-regulation of the cortisol and GH responses, and the pro-inflammatory and pro-immune cytokines remained at a basal or low level, with an increase of TNF-α and IL-6 in association with a higher level of IL-10 in the late phase of natural infection. The associated trends of pro-inflammatory and anti-inflammatory cytokines together with the cortisol level demonstrate that a previous vaccination promotes an early immune responsiveness in pigs and a more efficient control of inflammation in the late phase of infection with a heterologous PRRSV isolate; both events could sustain clinical protection.     

Influence of ampicillin, ceftiofur, attenuated live PRRSV vaccine, and reduced dose Streptococcus suis exposure on disease associated with PRRSV and S. suis coinfection

The objective of this research was to evaluate the efficacy of two antimicrobials (ampicillin and ceftiofur), a modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine, and low dose exposure to Streptococcus suis on disease associated with PRRSV/S. suis coinfection. Fifty-six, crossbred, PRRSV-free pigs were weaned at 10-12 days of age and randomly assigned to five treatment groups. All pigs were inoculated with 2ml of 10(6.4) TCID50/ml of high virulence PRRSV isolate VR-2385 intranasally at 29-31 days of age (day 0 of the study) followed 7 days later by intranasal inoculation with 2ml of 10(8.9)colony forming units(CFU)/ml S. suis type 2 isolate ISU VDL #40634/94. Pigs in group 1 (n=10) served as untreated infected positive controls. Pigs in group 2 (n=12) were treated with 5.0 mg/kg ceftiofur hydrochloride intramuscularly (IM) on days 8, 11, and 14. Pigs in group 3 (n=11) were treated with 11 mg/kg ampicillin IM on days 8-10. Pigs in group 4 (n=12) were vaccinated 14 days prior to PRRSV challenge with a commercial modified-live PRRSV vaccine. Pigs in group 5 (n=11) were exposed to a 1:100 dilution of the S. suis challenge inoculum 19 days prior to S. suis challenge. Mortality was 80, 25, 82, 83, and 36% in groups 1-5, respectively. The reduced dose S. suis exposure had some residual virulence, evidenced by S. suis induced meningitis in two pigs after exposure. Treatment with ceftiofur hydrochloride and reduced dose exposure to S. suis were the only treatments which significantly (P<0.05) reduced mortality associated with PRRSV/S. suis coinfection, significantly (P<0.05) reduced recovery of S. suis from tissues at necropsy, and significantly (P<0.05) reduced the severity of gross lung lesions.     

Expression of inflammatory cytokine mRNA in lymphoid tissue from swine experimentally infected with Mycobacterium avium serovar 2

OBJECTIVE: To evaluate in situ expression of inflammatory cytokine mRNA in lymphoid tissue of swine experimentally infected with Mycobacterium avium serovar 2. ANIMALS: 7 noninfected pigs and 7 pigs infected with M. avium serovar 2. PROCEDURE: Expression of mRNA of inflammatory cytokines such as tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta IL-6, and IL-8 in formalin-fixed paraffin-embedded blocks of lymphoid tissue (lymph nodes and tonsil) of swine experimentally infected with M. avium serovar 2 was compared with that of noninfected pigs. Tissues were evaluated by use of morphologic localization of cytokine mRNA, using in situ hybridization at 160 days after inoculation. RESULTS: A noticeable increase in mRNA expression for TNFalpha and mild increases in mRNA expression of IL-8 and IL-1beta were detected in mandibular lymph nodes from infected swine, compared with noninfected swine. Mild increase in mRNA expression for 1L-6 also was observed in tonsils from infected swine. Cytokine mRNA was detected in macrophages and lymphocytes, primarily within cortical follicles and adjacent mantle zones. CONCLUSIONS AND CLINICAL RELEVANCE: Expression of mRNA for inflammatory cytokines was increased in lymphoid tissue of infected swine, possibly resulting from local factors on, or secreted by, M. avium. These results suggest that alterations in cytokine mRNA expression are important in the pathogenesis and clinical course of mycobacteriosis in swine. Modulation of the immune response by vaccines that selectively target cytokine expression and secretion in response to mycobacterial challenge may be effective in prevention of mycobacteriosis in swine.