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More effective vaccines against Toxoplasma gondii may contribute to the control of this pathogen that has major veterinary and public health significance. In this study, two recombinant plasmids pcDNA/TgSAG1 and pVAX/mIL-18 containing T. gondii SAG1 (TgSAG1) and murine cytokine interleukin-18 (IL-18) were evaluated for their ability to protect mice against T. gondii challenge. Mice were given two intramuscular immunizations 3 weeks apart, and challenged with T. gondii 3 weeks later. All animals vaccinated with pcDNA/TgSAG1 alone or with pVAX/mIL-18 developed specific anti-TLA (T. gondii lysate antigen) antibodies and specific lymphocyte proliferative responses. Co-injection of pVAX/mIL-18 significantly increased the production of IFN-γ and IL-2. Further, challenge experiments showed that co-immunization with pVAX/mIL-18 significantly (< 0.05) increased the survival rate (60%), compared with pcDNA/TgSAG1 alone (40%). Therefore, codelivery of the IL-18-secreting plasmid potentiates the induction and maintenance of the type 1 helper T-cell immune response and may be a potent strategy for enhancing the protective efficacy of vaccines against T. gondii.  相似文献   

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Paratuberculosis is a chronic infection of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Early stage MAP infection can be detected by measuring cell-mediated immune responses using the interferon gamma (IFN-γ) assay. Whole blood samples are cultured overnight with specific MAP antigens followed by quantification of IFN-γ by ELISA. It is recommended that the time interval from sampling to culture does not exceed eight hours but addition of the co-stimulating cytokine interleukin 12 (IL-12) or anti-IL-10 antibodies to culture have been demonstrated to enhance IFN-γ responses of cultures stimulated with Johnin purified protein derivative (PPDj). Here we examined the correlation of IFN-γ production in response to PPDj and 15 recombinant antigens in day-old blood samples from heifers 10-21 months of age from a MAP infected herd with addition of either recombinant bovine IL-12 or anti-bovine IL-10 antibody with IFN-γ production in sample day samples. IFN-γ responses of sample day samples showed high correlation with responses to some antigens in day-old samples with addition of IL-12 or anti-IL-10 antibodies to cultures, indicating that day-old protocols can be applied as an alternative to the conventional IFN-γ protocol. Immunogenicity of the novel antigens was generally low for day-old samples. The most promising antigen using the day-old protocol with addition of IL-12 was latency protein LATP-2 as correlations, immunogenicity and diagnostic specificity collectively was high. The latency protein LATP-1 was the most promising antigen in the day-old protocol with addition of anti-IL-10 antibodies.  相似文献   

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The expression of several cytokines in spleen, pharyngeal lymph nodes, lung and brain after different immunization procedures and a challenge with 5 × 109 CFU of Haemophilus parasuis was compared. Five groups of colostrum-deprived pigs were used: vaccinated with (I) a bacterin, (II) an outer-membrane-protein-vaccine, (III) a recombinant transferring-binding protein B, (IV) exposed to a total dose of 105 CFU, and (V) not previously immunized. All pigs in groups III and V died, while all animals in group I, most of group IV and half of group II survived until the end of the experiment. IL-1α was found in significantly higher levels (p < 0.05) in spleen, lymph nodes and brain of dead pigs, which could be explained by the major severity of lesions in these animals. However, IL-4, IL-10, TNF-α and IFN-γ were expressed in significantly higher levels by survivors (for all the four cytokines in lymph nodes; for IL-4, IL-10 and TNF-α in spleen; for IL-4, TNF-α and IFN-γ in lung, and only for TNF-α in brain), thus suggesting a role of these four cytokines in the adaptive response, which might contribute to protection against H. parasuis infection.  相似文献   

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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.  相似文献   

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We determined whether a major Japanese cedar pollen allergen (Cry j 1) conjugated with CpG oligodeoxynucleotide would enhance allergen-specific Th1 responses in mice. Cry j 1 conjugated with CpG (Cry j 1-CpG) induced IL-12 in the spleen cells of naïve mice. Cry j 1-CpG immunization of BALB/c mice suppressed anti-Cry j 1 IgE response and enhanced anti-Cry j 1 IgG2a to subsequent Cry j 1 and alum adjuvant injection. CD4+T cells isolated from the spleens in mice immunized with Cry j 1-CpG produced higher IFN-γ levels than did CD4+T cells obtained from mice as negative controls. Our results suggested that Cry j 1-CpG immunization can induce Cry j 1-specific Th1 immune responses, thereby inhibiting IgE response to the pollen allergen.  相似文献   

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The immune response induced by intradermal vaccination using a needle-less device was evaluated in conventional pigs in comparison with the more conventional intramuscular vaccination; to this purpose, vaccination against Aujeszky’s Disease (AD) was used as a model of antiviral immunity. Two groups of pigs (n = 10 each) were vaccinated 4 weeks apart respectively by the intramuscular (IM group) and intradermal route (ID group; needle-less I.D.A.L.® vaccinator) with an AD modified live virus. Ten pigs injected with the vaccine adjuvant only were kept as sham-vaccinated controls (C group).On blood samples collected at 0, 2, 4, 5, 6 and 7 weeks post-vaccination (PV) ADV-specific virus neutralizing (VN) antibodies, IFN-γ secreting cells (SC), lymphocyte subsets and IFN-γ gene expression in PBMC were evaluated.VN antibodies increased after the 1st vaccination and peaked after the 2nd vaccination in both vaccinated groups. Also IFN-γ SC reached maximum levels in both groups after administration of the booster dose. Pigs in the control group remained negative for both parameters throughout the study. Flow cytometry showed persistently higher levels of CD3−CD8α+ Natural Killer cells in both vaccinated pigs. The ID group showed an earlier and regulated activation characterized by an increase of cytotoxic CD8β+ T lymphocytes and CD25+ cells after the boosting dose. No statistically significant differences between treated and control groups were detected for memory CD4+CD8α+low T cells. Upregulation of IFN-γ gene expression in PBMC was detected in ID and IM pigs after both vaccine administrations, although at a different extent. Overall, the results showed that the intradermal vaccine delivery by a needle-less device can prime a strong humoral and cellular immune response comparable to that obtained by the intramuscular vaccination.  相似文献   

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A limitation to the widespread application of interferon gamma (IFN-γ) response assays has been logistical difficulties, as they must be performed within hours of blood collection. Detection of specific IFN-γ responses to Mycobacterium avium subspecies paratuberculosis (MAP) as part of the cell-mediated immune response of ruminants with Johne's disease could aid in diagnosis and control programs. In this study, a modified protocol was developed in which cultures were supplemented with interleukin (IL)-7, a survival factor required to maintain resting T cells, alone or in combination with IL-12 to potentiate IFN-γ responses and extend blood storage time. The combination of IL-7 and IL-12 was synergistic, giving IFN-γ responses greater than with IL-12 alone, for sheep blood stored up to 2 days. In a cohort of naturally infected sheep, the same number of animals was identified as test positive using the modified assay (performed after 2 days with IL-7/IL-12 supplementation) as the standard IFN-γ assay performed on the day of blood collection. The modified assay offered greatest advantage in the detection of early stages of paratuberculosis infection, for sheep with low grade and paucibacillary lesions, and at early time points post-infection. The potentiation protocol allowed for practical shipment of blood samples from farm to laboratory, extending permissible transit times and applicability of IFN-γ testing to detect Johne's disease.  相似文献   

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The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in vitro assay for a direct read-out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evaluate for less severe or immunocompromising infections. Here we investigated the performance of the assay when recombinant co-stimulatory cytokines IL-12 and/or IL-18 were added along with Ag or PBS to cultures of whole-blood from pigs infected with Lawsonia intracellularis. In pigs recovering from a natural infection, addition of rIL-12 or rIL-18 alone increased the Ag-specific IFN-γ release while addition of both cytokines resulted in increased IFN-γ release also in PBS cultures. In analyses after experimental infections with L. intracellularis, significant increased levels of Ag-specific IFN-γ production were measured in Ag+rIL-18 cultures from infected pigs compared to the background response in PBS+rIL-18 control samples (p<0.01) or to Ag+rIL-18 cultures from non-inoculated control pigs (p<0.05). Flow cytometry identified two lymphocyte subsets as the Ag-specific IFN-γ producers. The highest IFN-γ production was by CD4(+)CD8(+) cells while a more numerous population of CD4(-)CD8(+) cells produced lower amounts of IFN-γ in response to rIL-18 and L. intracellularis Ag.  相似文献   

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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。  相似文献   

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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.  相似文献   

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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.  相似文献   

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Paratuberculosis was diagnosed in a goat herd that participated in a sanitation program against Mycobacterium avium subsp. paratuberculosis. The aim of this study was to characterise the development of gamma interferon (IFN-γ) and antibody responses as well as the occurrence of faecal shedding. Faecal culture appeared surprisingly sensitive as about 18% and 40% of the goats were positive at 9 and 15-17 months of age, respectively, and shedding was often seen prior to peripheral immune responses. Peripheral IFN-γ responses were not related to protection as clinical and high shedding goats often had high responses. An IFN-γ response usually preceded a humoral response. However, positive antibody titers could sometimes be seen simultaneously with, and even prior to, IFN-γ responses. In conclusion, faecal culture appeared as sensitive as IFN-γ testing. Furthermore, the antibody ELISA and the IFN-γ assay may perform equally well in an infected herd if surveillance is conducted annually.  相似文献   

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Although intestinal epithelial cells (IECs) are continuously exposed to high densities of enteric bacteria, they are not highly responsive to microbe-associated molecular patterns (MAMPs). However, inflammatory cytokines such as interferon-γ (IFN-γ) are potentially capable of priming IECs to enhance responsiveness to MAMPs. In this study, we observed that heat-killed Vibrio cholerae (HKVC) and its lipopolysaccharide (LPS) poorly induced IL-8 production in a human IEC line, HT-29. However, both HKVC and the LPS showed a substantial induction of IL-8 production in IFN-γ-primed HT-29 cells. LPS-induced IL-8 production was proportional to the IFN-γ-priming period and LPS could not induce IL-8 production in the presence of polymyxin B. Moreover, LPS-induced IL-8 production in the IFN-γ-primed HT-29 cells was mediated through signaling pathways requiring p38 kinase and ERK, but not the JNK/SAPK pathway. Since deleted in malignant brain tumor 1 (DMBT1) is known to interact with and antagonize the action of LPS, we hypothesized that IFN-γ enhanced the responsiveness to LPS in HT-29 through down-regulation of DMBT1. We found that IFN-γ indeed attenuated DMBT1 expression at both the mRNA and protein levels in HT-29 cells. Conversely, when the cells were transfected with small interfering RNA to specifically silence DMBT1, IL-8 expression was augmented even in the absence of IFN-γ and the augmentation was further enhanced by treatment with V. cholerae LPS. Since IFN-γ is known to increase IFN-β expression in the IECs, we examined if IFN-β functioned similar to IFN-γ. Although IFN-β alone was able to induce IL-8 expression, it failed to render HT-29 cells responsive to V. cholerae LPS. In conclusion, our study suggests that IFN-γ primes IECs to become responsive to V. cholerae and its LPS by suppressing the expression of DMBT1.  相似文献   

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IntroductionCystic echinococcosis (CE) is a chronic zoonotic disease caused by the larval stage of Echinococcus granulosus (E. granulosus), which affects domestic and wild carnivores as the definitive host and ungulates as intermediate hosts. In intermediate hosts, both Th1 and Th2 cells are involved in the immune responses to an echinoccocal infection. This study aimed to investigate production of IL-4, IL-10, and IFN-γ cytokines in peripheral blood mononuclear cells (PBMCs) of CE patients before and after surgical treatment.MethodsTo evaluate cytokine production in response to E. granulosus antigens, we investigated IL-4, IL-10, and IFN-γ production in PBMCs of 20 CE patients in response to hydatid cyst fluid antigen (HCF-Ag) before and after surgical treatment using ELISA.ResultsThe mean IL-4 production from HCF-Ag stimulated PBMCs was significantly decreased (p < 0.05), while IFN-γ was significantly increased in HCF-Ag stimulated PBMCs in patients after surgery (p = 0.005).Furthermore, our results showed that there is no significant difference between IL-10 production in patients before and after treatment (p = 0.562).ConclusionsOur data Indicated production of IL-4 in cultured PBMCs of CE patients stimulated with HCF-Ag was decreased significantly. While, production of IFN-γ was increased significantly in responses to HCF Ag after surgery. We concluded that the evaluation of IL-4 and IFN-γ in HCF-Ag stimulated PBMCs of CE patients should be considered as a useful marker in the follow up of patients with cystic echinococcosis.  相似文献   

18.
To explore the potential of a novel animal interferon formulation for controlled release, the yak interferon-alpha (IFN-α) glutathione S-transferase (GST) fusion protein was expressed in Escherichia coli (E. coli) and the purified recombinant IFN-α was encapsulated into solid lipid nanoparticles (SLN) by double emulsion solvent evaporation (w/o/w) method. The particle size and zeta potential of IFN-α-loaded SLN were 124.2 ± 10.2 nm and −11.2 ± 0.6 mV. The encapsulation efficiency of IFN-α and loading capacity of the SLN were 83.7 ± 4.5% and 1.73 ± 0.15%, respectively. In vitro release study and antiviral assay demonstrated that the IFN-α released from the SLN in a 16-day period exhibited antiviral activity in Madin-Darby bovine kidney (MDBK) cells against vesicular stomatitis virus (VSV), and showed a release pattern of an initial burst release followed by a sustained and slow release. Cytotoxicity assay in cell culture demonstrated that the SLN were not toxic. The results of this exploratory study suggest that the IFN-α-loaded SLN could be a useful formulation for controlled release in veterinary therapeutics.  相似文献   

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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.  相似文献   

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本试验旨在比较党参多糖(CP)与硫酸化党参多糖(SCP)对H2O2诱导RAW264.7细胞急性损伤及小鼠急性酒精损伤的保护作用。采用H2O2诱导RAW264.7细胞急性损伤,检测不同浓度CP与SCP对RAW264.7细胞生长、吞噬功能及IL-2、IL-4、IL-10和IFN-γ水平的影响。随后体内建立小鼠急性酒精损伤模型,将90只4周龄雄性昆明小鼠随机均分为9组:正常对照组(B)、模型对照组(MO)、维生素C对照组(VC)、3个剂量的CP(CPL、CPM、CPH)与SCP(SCPL、SCPM、SCPH)组。检测各组对急性酒精损伤小鼠生长情况、血液生理指标、脏器指数、血清IL-2、IL-4、IL-10及IFN-γ水平的影响。结果显示,H2O2诱导RAW264.7细胞急性损伤后,与MO组相比,CP和SCP组细胞贴壁数量增加,细胞密度增大,37 ℃作用30、60、90 min后均能显著提高细胞吞噬能力(P < 0.05),且SCP各组促进细胞增殖及吞噬作用优于CP各组。CP和SCP均能显著促进细胞分泌IL-2、IFN-γ(P < 0.05),抑制IL-4的分泌(P < 0.05),SCP各组对IL-4的抑制作用优于CP,其中SCPL组对IL-4的抑制效果最佳。小鼠急性酒精损伤模型中,与MO组相比,CP和SCP能显著改善小鼠血液生理指标(P < 0.05),使血液中WBC和HGB恢复至正常水平,其中SCPL组效果最佳。CP和SCP还能改善小鼠血生化指标,显著提高血清中TC、LDH含量(P < 0.05),降低TG含量(P < 0.05)。CP和SCP组较MO组小鼠血清中IL-2、IFN-γ水平显著升高(P < 0.05),IL-4水平显著降低(P < 0.05),其中SCP各组对IL-4的抑制作用最佳。综上所述,SCP可显著提高H2O2诱导RAW264.7细胞急性损伤后细胞吞噬能力,促进细胞分泌IL-2、IFN-γ水平,抑制IL-4分泌;改善酒精诱导的急性损伤小鼠的血液生理指标、血清生化指标和免疫因子,具有一定的保护作用,且SCP在一定条件下效果优于CP。  相似文献   

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