All three classes of CpG ODNs up-regulate IP-10 gene in pigs

The analysis of CpG ODN induced innate immune responses in different animal species has shown substantial similarities and differences in levels and types of induced cytokines profile. The objectives of these studies were to identify innate immune biomarkers activated by three classes of CpG ODNs in pigs. For this purpose, we investigated the kinetics of innate immune responses in immune cells from pigs following in vitro and in vivo stimulation with CpG ODNs. The mRNA expression of cytokine and chemokine genes were assayed by SYBR^@ green based quantitative real time PCR. A-class CpG ODN induced significant but transient levels of IFN-γ, IL-12 (P40), IL-6, IL-4 and TNF-α mRNA, C-class CpG ODN induced significant level of IFN-γ, IFN-α and IL-12 mRNA and the lowest level of IL-4 (Th-2 type) mRNA. A very low level of some cytokines stimulation was observed by GC ODNs. It is noteworthy, that IL-12 (P35) mRNA was significantly stimulated by B-class GpC ODN 7909. Interestingly, all classes of CpG ODNs induced significant level of IP-10 at 12 h post stimulation. These in vitro and in vivo observations suggest that interferon-γ inducible protein 10 (IP-10) may be a reliable biomarker for immune activity induced by CpG ODNs in pigs.     

Blood cellular immune response in pigs immunized and challenged with Haemophilus parasuis

The cellular immune response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer’s disease in pigs, was characterized studying changes in peripheral blood mononuclear cells (PBMC) in colostrum-deprived pigs. Five groups were studied, four of those were previously immunized with different formulations and the fifth was maintained as non-immunized control. All groups were challenged with 5 × 10⁹ CFU of H. parasuis serotype 5. The non-commercial bacterin conferred a complete protection, while the OMP-vaccine and the exposure to a subletal dose of 10⁵ CFU of H. parasuis protected only partially, and the recombinant Tbp B-vaccine induced no protection. PBMC were analyzed using monoclonal antibodies against porcine CD45⁺, CD3⁺, CD4⁺, CD8α⁺, CD25⁺, CD4⁺ naïve, αIgM⁺ and SWC3⁺ cells in single-colour fluorescence, and CD4⁺/CD8α⁺ and CD8α⁺/CD8β⁺ combinations in two-colour fluorescence. The different groups showed no significant changes in PBMC subsets following vaccination, and only minor changes were encountered after challenge, consisting mainly of significant increases (P < 0.05) in the relative proportions of monocytes and granulocytes (SWC3⁺) and B cells (αIgM⁺), as well as a significant reduction in CD3⁺ cells (P < 0.05). These changes were similar for the five groups compared, except for the significant increase of CD25⁺ cells, which was only observed for the bacterin-vaccinated group. These results suggest an increase of trafficking of inflammatory cells and the onset of the adaptive antibody response against H. parasuis infection; in addition, the blood cellular response developed by the different groups was not relevant to protection.     

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 antibody response in colostrum-deprived pigs experimentally infected with Haemophilus parasuis

The serum antibody response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer’s disease in pigs, was characterized by ELISA measuring IgM and IgGt levels against whole-cells and outer-membrane-proteins (OMPs) as antigens. Five groups of pigs were studied, four of those were previously immunized with different formulations, and the fifth was maintained as non-immunized control. All groups were challenged with 5 × 10⁹ CFU of H. parasuis. The non-commercial bacterin induced a full protection against disease, the OMP-vaccine and the exposure to a sublethal dose of 10⁵ CFU protected only partially, and the recombinant TbpB-vaccine conferred no protection. The humoral response in the pigs that died after infection (all controls, all those vaccinated with the recombinant TbpB, and two of both those inoculated with OMPs and those exposed to the sublethal dose) could be only measured before it, but it was irrelevant in all cases. However, a specific IgM and IgGt production was observed before challenge in all the surviving pigs, irrespective of the type of immunization received. This antibody response was even greater after H. parasuis infection, especially in those survivors receiving the sublethal dose. These results suggest a role of the antibodies developed after the different immunization protocols in preventing infection and death; therefore, the humoral immunity is protective against experimental Glässer’s disease.     

Growth hormones therapy in immune response against Trypanosoma cruzi

Growth hormone (GH) is an important hypophyseal hormone that is primarily involved in body growth and metabolism. In mammals, control of Trypanosoma cruzi parasitism during the acute phase of infection is considered to be critically dependent on direct macrophage activation by cytokines. To explore the possibility that GH might be effective in the treatment of Chagas’ disease, we investigated its effects on the course of T. cruzi infection in rats, focusing our analyses on its influences on parasitemia, NO, TNF-α and IFN-γ concentration and on histopathological alterations and parasite burden in heart tissue. T. cruzi-infected male Wistar rats were intraperitoneally treated with 5 ng/10 g body weight/day of GH. Animals treated with GH showed a significant reduction in the number of blood trypomastigotes during the acute phase of infection compared with untreated animals (P < 0.05). For all experimental days (7, 14 and 21 post infection) of the acute phase, infected and GH treated animals reached higher concentrations of TNF-α, IFN-γ and nitric oxide as compared to untreated and infected counterparts (P < 0.05) Histopathological observations of heart tissue revealed that GH administration also resulted in fewer and smaller amastigote burdens, and less inflammatory infiltrate and tissue disorganization, indicating a reduced parasitism of this tissue. These results show that GH can be considered as an immunomodulator substance for controlling parasite replication and combined with the current drug used may represent in the future a new therapeutic tool to reduce the harmful effects of Chagas’ disease.     

Role of IFN-α during the acute stage of a swine influenza virus infection

Cytokines, especially interferon-alpha (IFN-α) are important in controlling influenza virus infections. To investigate the role of IFN-α in influenza, the swine IFN-α neutralizing monoclonal antibody (Ab) K9 was applied in a swine model of influenza A virus infection. First, the optimal dose and route for administration of the IFN-α neutralizing Abs was determined. Based on those results, the effect of the Abs on a swine influenza virus infection was investigated. Pigs were inoculated intratracheally with 10^6.0 mean egg infectious dose (EID₅₀) A/Swine/Belgium/1/98 (H1N1) virus. At the time of challenge and 18 h later, they were injected intratracheally and intraperitoneally with a high dose of IFN-α neutralizing Abs or control Abs. The animals were euthanized at 0, 24, 30, 48 and 72 h after inoculation. At 24 and 30 h, IFN-α levels in broncho-alveolar lavage fluid of K9 recipient animals were strongly suppressed, and this coincided with reduced IL-6 and IL-12 levels. TNF-α and IL-1 levels were unaffected compared to those in the control Ab treated group. Importantly, the onset and peak of clinical symptoms in IFN-α neutralizing Abs treated animals were delayed by 24 h, simultaneously with the suppression of IFN-α, but there was no obvious effect on virus replication and lung pathology. These results suggest an important role for IFN-α in IL-6 and IL-12 induction and a role of all three cytokines in the symptoms of swine influenza.     

Intratesticular expression of mRNAs of both interferon γ and tumor necrosis factor α is significantly increased in experimental autoimmune orchitis in mice

Experimental autoimmune orchitis (EAO) is one of the models of immunological male infertility. Murine EAO is CD4+T cell-dependent and classically induced by immunization with a testicular homogenate and adjuvants. We previously established that immunization with viable syngeneic testicular germ cells (TGC) can also induce murine EAO with no use of any adjuvant. Analyses of this EAO model have already revealed that cultured spleen cells of immunized mice secreted interferon (IFN)-γ and that treatment of the immunized mice with anti-IFN-γ monoclonal antibodies significantly suppressed the EAO. It is known that both IFN-γ and tumor necrosis factor (TNF)-α are representative cytokines of Th1 cells and exhibit local toxicity toward the seminiferous epithelium in vivo. However, changes in these two cytokines in EAO-affected testes have not yet been investigated. Therefore, in the present study, we investigated the expression of intratesticular IFN-γ and TNF- α mRNAs in TGC-induced EAO using real-time RT-PCR. The results demonstrated that the intratesticular mRNAs for both IFN-γ and TNF-α significantly increased, while other cytokines such as IL-1α, IL-1β, IL-6 and TGF-β did not show dramatic changes in the immunized mice. These results suggest that secretion of significant amounts of IFN-γ and TNF-α in situ contributes to the spermatogenic disturbance in EAO.     

Enhanced proinflammatory cytokine activity during experimental bluetongue virus-1 infection in Indian native sheep

Bluetongue disease has been causing variable morbidity and mortality in sheep in India and other countries of the world. The experimental infection with Indian BTV-1 strain induced mild to sub-acute infection in native sheep. There were low induction of IL-12, IFN-γ and TNF-α cytokine mRNA expressions determined by real time RT-PCR in draining lymph nodes (DLN), spleen, and PBMCs during the initial stages of infection (8 days post inoculation, DPI) and higher around 15 DPI. The reduced pro-inflammatory cytokine (IFN-γ and TNF-α) responses during the initial stage of infection (8 DPI) was also accompanied by similarly decreased T-cell populations and overt clinical symptoms. Later up regulation of these cytokines and substantial increase in the proportion of CD8(+) T-cells occurred with reduction of clinical signs and disappearance of BTV-1 antigen from tissues as determined by immunohistochemistry and RT-PCR. Thus there is definite involvement of pro-inflammatory cytokines and CD8(+) T cell activity in disease induced by BTV-1 strain.     

人参皂苷-Rh2衍生物体外诱生小鼠细胞因子的作用及对NO水平的影响

探讨了经分子修饰后的人参皂苷-Rh2(衍生物)对小鼠脾T淋巴细胞分泌IL-2与IFNγ-、腹腔巨噬细胞分泌TNF-α与NO水平的影响。应用双抗体夹心ELISA法测定3种细胞因子的含量,硝酸还原酶法测定NO的含量。结果表明,衍生物的3个浓度(100,200和300μg/mL)均抑制了IL-2(P<0.01)和NO(P>0.05)的分泌,明显促进了IFN-γ和TNF-α的分泌(P<0.01)。提示衍生物可能是通过细胞因子途径来调节机体的免疫功能。     

Cytokines and acute phase proteins associated with acute swine influenza infection in pigs

This study set out to investigate the cytokines and acute phase proteins (APPs) associated with the acute stages of experimentally-induced swine influenza virus (SIV) infection in 3-week-old, colostrum-deprived, caesarean-derived piglets. The piglets were inoculated intratracheally with 10^7.5 50% egg infective dose [EID₅₀] Swine/Belgium/1/98 (H1N1) SIV and were euthanased at time-points between 0 and 120 h post-inoculation (PI). Broncho-alveolar lavage fluid (BALF), lung homogenates and sera were examined for inflammatory mediators by bioassay or ELISA. Interferon (IFN)-α, interleukin (IL)-6, IL-1 and tumour necrosis factor (TNF)-α peaked in BALF 24–30 h PI, when virus titres and the severity of clinical signs were maximal.Whereas IFN-γ and IL-12, but not IL-18, increased in tandem in BALF, serum cytokine concentrations were either undetectable or were up to 100-fold lower. The APP C-reactive protein (CRP) and haptoglobin peaked 24 h later than the cytokines and reached higher levels in serum than in BALF. In contrast, lipopolysaccharide (LPS)-binding protein (LBP) only increased in BALF. Lung virus titres tightly correlated with BALF IFN-α, IL-6, IL-1, TNF-α, IFN-γ and IL-12, as well as with serum IL-6, IFN-α and IFN-γ. Signs of disease correlated with the same cytokines in BALF and serum, as well as with BALF LBP and serum CRP. The findings suggest that IFN-γ and IL-12 play a role in the pathogenesis of SIV and that APPs are induced by cytokines. This influenza infection model may have value in assessing the therapeutic potential of cytokine antagonists.     

Differential expression of pro-inflammatory and anti-inflammatory cytokines during experimental infection with low or high virulence bovine viral diarrhea virus in beef calves

The objective was to compare the mRNA expression of pro-inflammatory (TNF-α, IL-1β, IFN-γ, IL-2, IL-12, IL-15) and anti-inflammatory (IL-4, IL-10, TGF-β) cytokines, after experimental infection with low or high virulence noncytopathic (ncp) bovine viral diarrhea virus (BVDV). Thirty BVDV-naïve, beef calves were intranasally inoculated with low (LV; n = 10, SD-1) or high (HV; n = 10, 1373) virulence ncp BVDV or with BVDV-free cell culture medium (Control, n = 10). Calves were euthanized on day 5 post-inoculation, and tracheo-bronchial lymph node and spleen samples were collected for mRNA expression through quantitative-RT-PCR. mRNA levels of pro-inflammatory (TNF-α, IL-1β, IL-2, IFN-γ) and anti-inflammatory (IL-4 and IL-10) cytokines were up-regulated in tracheo-bronchial lymph nodes of HV, but not in LV, compared to the control group (P < 0.05). IL-12 mRNA level was up-regulated in tracheo-bronchial lymph nodes of both LV and HV groups (P ≤ 0.05). A significant up-regulation of IL-15 mRNA was observed in tracheo-bronchial lymph nodes for LV calves (P < 0.002), but not for HV calves. Experimental inoculation with BVDV-2 1373 stimulated significant mRNA expression of pro-inflammatory and anti-inflammatory cytokines. In contrast, inoculation with BVDV-1a SD-1 only resulted in up-regulation of IL-12 and IL-15 mRNA, which is associated with activation of macrophages and NK cells during innate immune response.     

Inflammation-associated responses in piglets induced with post-weaning colibacillosis are influenced by dietary protein level

Forty piglets (average body weight = 5.32 kg) were used to investigate the effect of dietary crude protein (CP) content on immunological responses following a challenge with enterotoxigenic Escherichia coli (ETEC) K88. Pigs, housed 4 per pen, were randomly allotted to 2 diets: 1) a high, 225 g/kg CP diet (HCP) or 2) a low, 176 g/kg CP diet (LCP) supplemented with crystalline amino acids. Pigs were orally challenged with 6 mL of an ETEC K88 suspension containing 10¹⁰ cfu/mL on d 8 after weaning. Blood samples were collected from 10 pigs (1 pig/pen) on d 7 (at weaning), −24 h, 8 h, 72 h and 7 d after the challenge for determination of plasma urea N (PUN) and serum concentrations of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) and haptoglobin (Hp). Tumor necrosis factor alpha, IL-1β and Hp were measured as indicators of inflammatory responses. The concentrations of serum TNF-α at 8 h, 72 h and 7 d after challenge were similar to the level observed at 24 h before challenge but higher (P < 0.05) than the weaning level. Pigs fed the LCP diet had lower (P = 0.032) concentrations of IL-1β (72 vs. 116 pg/mL) at 8 h post-challenge compared with those fed the HCP diet. Likewise, pigs fed the LCP diet tended to have lower (P = 0.088) concentration of Hp (9 vs. 25 mg/dL) compared with those fed the HCP diet at 8 h post-challenge. Compared with the weaning concentration, PUN concentration at 72 h after ETEC challenge was higher (P < 0.05) in pigs fed the HCP diet. The results indicate that the LCP diet supplemented with crystalline amino acids reduced inflammatory responses, as indicated by serum IL-1β, in piglets infected with ETEC K88.     

地锦草总黄酮对小鼠免疫功能及细胞因子mRNA表达影响

探讨地锦草总黄酮对小鼠免疫功能及细胞因子IL-2、IL-12、IFN-γ、TNF-αmRNA表达影响。将小鼠随机分成4组：地锦草总黄酮高剂量组（H组）、中剂量组（M组）、低剂量组（L组）和纯水对照组（C组）,连续灌胃9 d后,检测其单核巨噬细胞吞噬功能、2%绵羊红细胞诱导的小鼠迟发型变态反应、肝脏指数和脾脏指数,并用逆转录酶-聚合酶链式反应的方法检测脾脏IL-2、IL-12、IFN-γ和TNF-αmRNA表达水平。结果显示,地锦草总黄酮3个剂量组脾脏指数、廓清指数（K）、吞噬指数（α）、24 h足跖增厚值均显著高于C组;M组和H组肝指数显著高于C组（P〈0.01）;3个剂量组均能提高IL-2、IL-12、IFN-γ和TNF-αmRNA的表达水平,与C组相比,H组最显著。试验结果表明,地锦草总黄酮能有效提高机体的免疫功能及IL-2、IL-12、IFN-γ和TNF-αmRNA表达。     

3种猪细胞因子mRNA实时荧光定量RT-PCR检测方法的建立与应用

为建立猪细胞因子SYBR Green Ⅰ实时荧光定量RT-PCR检测方法,根据GenBank中3种重要的猪细胞因子即猪白细胞介素-2(interleukin-2,IL-2)、α-干扰素(interferon α,IFN-α)和肿瘤坏死因子-α(tumor necrosis factor,TNF-α)的基因序列,设计特异引物扩增目的基因.将3种基因克隆至pMD18-T载体上,得到各自阳性克隆质粒,以3种阳性质粒为标准品建立标准曲线并进行熔解曲线分析以及灵敏性、特异性和重复性试验.结果表明,当标准品稀释度为1×10¹~1×10⁶ 拷贝/μL时,3种基因的Ct值与浓度间具有良好的线性关系,相关系数均≥0.992.熔解曲线分析表明,产物为特异性单峰且重复性较好.应用建立的方法对猪繁殖与呼吸综合征病毒(PRRSV)TJM-F92株免疫的30日龄猪外周血单核细胞(PBMC)中IL-12、IFN-α和TNF-α表达量进行检测,结果发现,免疫了PRRSV TJM-F92株的猪PBMC细胞内3种细胞因子表达量均极显著升高(P< 0.01).研究结果为IL-12、IFN-α和TNF-α的定量分析提供了技术平台.     

Constitutive expression of interferons in swine leukocytes

Interferon (IFN)-α and IFN-γ positive cells were revealed by flow cytometry in peripheral blood mononuclear cells (PBMC) of Specific Pathogen Free (SPF) pigs. A low prevalence of IFN-γ positive cells was also detected in PBMC of some Porcine Reproductive and Respiratory Syndrome virus-infected pigs and uninfected, control pigs. IFN-α positive cells showed phenotypes of both monocytes and plasmacytoid dendritic cells. The presence of IFN-α in PBMC was also confirmed by Western blotting. By immunoprecipitation, IFN-α was detected as 32 and 55-57 kDa bands in PBMC of healthy SPF piglets. These samples were also IFN-γ positive; the cytokine was revealed as 24, 37 and 54 kDa bands. The unusual molecular mass values of intracellular interferons were probably due to oligomerization, as previously described for human IFN-α. Swine intracellular IFN-α displayed the expected antiviral activity on bovine MDBK cells. The results indicate that interferons are constitutively expressed in swine leukocytes with peculiar molecular features.     

Alteration in lymphocyte responses,cytokine and chemokine profiles in chickens infected with genotype VII and VIII velogenic Newcastle disease virus

Newcastle disease (ND) is a highly contagious avian disease and one of the major causes of economic losses in the poultry industry. The emergence of virulent NDV genotypes and repeated outbreaks of NDV in vaccinated chickens have raised the need for fundamental studies on the virus–host interactions. In this study, the profiles of B and T lymphocytes and macrophages and differential expression of 26 immune-related genes in the spleen of specific-pathogen-free (SPF) chickens, infected with either the velogenic genotype VII NDV strain IBS002 or the genotype VIII NDV strain AF2240, were evaluated. A significant reduction in T lymphocyte population and an increase in the infiltration of IgM⁺ B cells and KUL01⁺ macrophages were detected in the infected spleens at 1, 3 and 4 days post-infection (dpi) (P < 0.05). The gene expression profiles showed an up-regulation of CCLi3, CXCLi1, CXCLi2 (IL-8), IFN-γ, IL-12α, IL-18, IL-1β, IL-6, iNOS, TLR7, MHCI, IL-17F and TNFSF13B (P < 0.05). However, these two genotypes showed different cytokine expression patterns and viral load. IBS002 showed higher viral load than AF2240 in spleen at 3 and 4 dpi and caused a more rapid up-regulation of CXCLi2, IFN-γ, IL-12α, IL-18, IL-1β, iNOS and IL-10 at 3 dpi. Meanwhile, the expression levels of CCLI3, CXCLi1, IFN-γ, IL-12α, IL-1β and iNOS genes were significantly higher in AF2240 at 4 dpi. In addition, the expression levels of IL-10 were significantly higher in the IBS002-infected chickens at 3 and 4 dpi. Hence, infection with velogenic genotype VII and VIII NDV induced different viral load and production of cytokines and chemokines associated with inflammatory reactions.     

Evaluation on the pathogenicity of Erysipelothrix tonsillarum for pigs by immunosuppression with cyclophosphamide or dexamethasone

The pathogenicity of Erysipelothrix tonsillarum was evaluated in pigs immunosuppressed with cyclophosphamide (CY) or dexamethasone (DM). Animals were treated with 15 mg/kg CY (n = 8, five injections), 1 mg/kg DM (n = 8, nine injections) or left untreated (n = 8). On the fifth day after the beginning of drug treatments, swine were inoculated with one of two E. tonsillarum serovar 7 strains (approximately 10⁶ CFU per pig). In the CY-treated group, both circulating neutrophil and lymphocyte counts decreased, whereas in the DM-treated group, lymphocyte counts decreased but neutrophil counts increased. During the observation period, none of the CY- or DM-treated pigs developed clinical signs or gross lesions, as well as non-treated pigs. Growth agglutination antibody titres in all pigs remained unchanged. Our findings indicate that E. tonsillarum strains are avirulent for swine, regardless of immune status.     

Cultivation, LD(50) determination and experimental model of Streptococcus suis serotype 2 strain HA9801

The effects of nutritional components and submerged culture conditions on colony-forming unit (CFU) counts by Streptococcus suis serotype 2 strain HA9801 in flask culture was investigated, and the optimal medium and cultivation conditions was confirmed by using a 50 l bioreactor. The LD₅₀ values of HA9801 in pigs before and after fermentation were 1.8 × 10⁷ CFU, which indicated that the virulence of HA9801 was very stable in the fermentation process. In addition, an experimental model that closely mimics naturally occurring disease in conventional pigs was established.     

Adjuvant effect enhancement of porcine interleukin-2 packaged into solid lipid nanoparticles

In this paper, we investigated the enhancement of adjuvant effects of porcine IL-2 (pIL-2) by packaging it into a solid lipid nanoparticle (SLN) delivery system. SLN–pIL-2 was prepared using hydrogenated castor oil and Polylactide-co-glycolide by double emulsion solvent evaporation methods (w/o/w). In animal trials, BALB/c mice were immunized with inactivated foot and mouth disease virus (FMDV) antigen combined with the SLN–pIL-2 adjuvant on days 0 and 14. Antibody titer, splenocyte proliferation, and secretion of IFN-γ and IL-4 cytokines were determined. Our results showed that SLN–pIL-2 could significantly enhance FMDV-specific antibody level compared with recombinant pIL-2 alone (p < 0.05). In addition, SLN–pIL-2 significantly increased the proliferative responses of antigen-specific spleen cells. Furthermore, SLN–pIL-2 induced the secretion of IFN-γ at a level higher than that induced by recombinant pIL-2 alone. Our results indicate that packaging recombinant pIL-2 in SLNs can be an effective way of boosting the effectiveness of pIL-2 as an adjuvant to enhance immune responses of vaccines.