Characterization of the invasion of porcine endothelial cells by Streptococcus suis serotype 2

Streptococcus suis serotype 2 is an important swine pathogen associated mainly with meningitis. In a previous study, we demonstrated the ability of S. suis serotype 2 to adhere to and invade immortalized porcine brain microvascular endothelial cells (PBMECs) forming the blood-brain barrier. The aim of the current work was to further characterize the mechanism(s) by which S. suis invades porcine endothelial cells. The ability of several S. suis strains to interact with PBMECs was not found to correlate with their geographic origin, virulence, host of origin, or suilysin production. Characterization studies demonstrated that proteinaceous adhesins/invasins, cell wall components, lipoteichoic acid, and serum components (including fibronectin) were involved in interactions between S. suis and PBMECs. In addition to PBMECs, S. suis was able to adhere to and invade 2 porcine aortic endothelial cell lines and primary PBMECs.     

人端粒酶催化亚基和SV40大T抗原永生化猪脐静脉血管内皮细胞

为建立稳定的猪血管内皮细胞系,用于猪瘟病毒(CSFV)的致病机理研究提供理想的细胞模型,本研究利用逆转录病毒分别转导人端粒酶催化亚基(hTERT)基因或SV40大T抗原(SV40 LT)基因进入猪脐静脉血管内皮细胞(SUVEC),通过G418或嘌呤霉素筛选阳性克隆,并进行细胞传代研究和细胞形态学及表型鉴定。分别获得了两种方法建立的永生化猪血管内皮细胞系SUVEC-hT和SUVEC-ST。两种细胞系均呈铺路石样单层排列生长,具有高度的增殖活性,在传代70代后不表现任何衰老细胞的特征,并保持接触生长抑制。SUVEC-hT细胞系持续表达hTERT、CD31、CD34和von Willebrand factor,并能摄取Dil标记的乙酰化低密度脂蛋白(Dil-Ac-LDL)。SUVEC-ST细胞系持续表达SV40 LT、CD31和CD34,并能摄取Dil-Ac-LDL。两个细胞系均保持正常的核型并对CSFV敏感。结果表明通过表达外源的hTERT或SV40 LT,SUVEC已被永生化,并且保留了血管内皮细胞的主要生物学特征。     

Porcine endogenous retroviruses (PERV): in vitro artifact or a big problem for xenotransplantation?]

The pig is the favorized donor species for clinical xenotransplantation. However, PATIENCE et al. could show, that porcine endogenous retroviruses (PERV), released by a porcine kidney cell line, are capable of infecting human cell lines in vitro. Based on this discovery there is an ongoing discussion concerning the risks of zoonosis combined with xenotranplantation, which culminated in the demand for a moratorium on clinical transplantation of porcine organs. Recent findings exclude the possibility of an artifact due to the use of an immortalized cell line: Release of infectious PERV was also shown for mitogenic stimulated primary porcine peripheral blood mononuclear cells and, even more important, for primary porcine endothelial cells. In contrast, none of the recent retrospective in vivo studies showed evidence for PERV transmission, neither in patients after transplantation of porcine pancreas islet cells or after extracorporal perfusion of porcine kidneys, nor in baboons after transplantation of porcine endothelial cells. Currently it is not known, whether impairments of the immunological responses against foreign pathogens, which are associated with different xenotransplantation strategies, could enable PERV in vivo infection. Only in vivo experiments, if possible in suitable subhuman primate models, offer the prospect for a final risk assessment.     

In vitro effect of classical swine fever virus on a porcine aortic endothelial cell line

The effect of classical swine fever (CSF) virus on some phenotypic and functional features of an established porcine aortic endothelial cell (AOC) line was investigated. AOC cells show most of the characteristics of primary endothelial cells, avoiding the alterations and senescence that these cells undergo after a few passages in culture. AOC cells were susceptible to CSF virus infection to a high degree, reaching 90% of CSF virus positive cells after 24 h of infection; however as with other porcine susceptible cells, no cytopathic effect could be observed. In these conditions none of the surface molecules studied, including SLA-II MHC antigens, adhesion or co-stimulatory molecules, were altered by virus infection after 24 or 48 h. Functionally CSF virus infection induced a decrease in the pro-coagulant activity of the AOC cells, determined by the increase in the clot formation time shown by the lysates of these cells. This contrasts with the increase observed in the expression of mRNA corresponding to IL-1 alpha and IL-6, two proinflammatory and pro-coagulant cytokines, in CSF virus-infected AOC cells.     

Correlation of cell surface marker expression with African swine fever virus infection

The expression of surface markers on African swine fever virus (ASFV) infected cells was evaluated to assess their involvement in infection. Previous findings indicated CD163 expression was correlated with ASFV susceptibility. However, in this study the expression of porcine CD163 on cell lines did not increase the infection rate of these cells indicating other factors are likely to be important in determining susceptibility to infection. On adherent porcine bone marrow (pBM) cells the expression of CD45 was strongly correlated with infection. CD163 and CD203a expression correlated at intermediate levels with infection, indicating cells expressing these markers could become infected but were not preferentially infected by the virus. Most of the cells expressing MHCII were infected, indicating that they may be preferentially infected although expression of MHCII was not essential for infection and a large percentage of the infected cells were MHCII negative. CD16 showed a marked decrease in expression following infection and significantly lower levels of infected cells were shown to express CD16. Altogether these results suggest CD163 may be involved in ASFV infection but it may not be essential; the results also highlight the importance of other cell markers which requiring further investigation.     

Membrane markers of the immune cells in swine: an update

Besides their breeding value, swine are increasingly used as biomedical models. As reported in three international swine clusters of differentiation (CD) workshops and in the animal homologue section of the last workshop for the determination of human leukocyte differentiation antigens (HLDA 8), characterisation of leukocyte surface antigens by monoclonal antibodies and other molecular studies have determined the cell lineages and blood leukocyte subsets implicated in the immune response, including cell adhesion molecules involved in cell trafficking. This review focusses on the current state of knowledge of porcine leukocyte differentiation and major histocompatibility complex (SLA) molecules. Examples of porcine particularities such as the double-positive T lymphocytes with the phenotype CD(4+)CD8(low) and CD(4-)CD8(low) alphabeta T cell subsets and the persistence of SLA class II after T-lymphocyte activation are illustrated, as well as the shared characteristics of the Artiodactyla group, such as the high proportion of gammadelta TcR (T cell receptor) T cells in blood and other lymphoid tissues. Furthermore, discrepancies between swine and humans, such as CD16 expression on dendritic cells and CD11b (wCD11R1) tissue distribution are outlined. The rapidly growing information should facilitate manipulation of the swine immune system towards improving disease control, and open new avenues for biomedical research using the pig as a model.     

Susceptibility of primary human endothelial cells to C. perfringens beta-toxin suggesting similar pathogenesis in human and porcine necrotizing enteritis

Clostridium perfringens type C causes fatal necrotizing enteritis in different mammalian hosts, most commonly in newborn piglets. Human cases are rare, but the disease, also called pigbel, was endemic in the Highlands of Papua New Guinea. Lesions in piglets and humans are very similar and characterized by segmental necro-hemorrhagic enteritis in acute cases and fibrino-necrotizing enteritis in subacute cases. Histologically, deep mucosal necrosis accompanied by vascular thrombosis and necrosis was consistently reported in naturally affected pigs and humans. This suggests common pathogenetic mechanisms. Previous in vitro studies using primary porcine aortic endothelial cells suggested that beta-toxin (CPB) induced endothelial damage contributes to the pathogenesis of C. perfringens type C enteritis in pigs. In the present study we investigated toxic effects of CPB on cultured primary human macro- and microvascular endothelial cells. In vitro, these cells were highly sensitive to CPB and reacted with similar cytopathic and cytotoxic effects as porcine endothelial cells. Our results indicate that porcine and human cell culture based in vitro models represent valuable tools to investigate the pathogenesis of this bacterial disease in animals and humans.     

2019年猪病流行情况与2020年流行趋势及防控对策

文章结合实验室的监测数据,概述了2019年我国非洲猪瘟、猪繁殖与呼吸综合征、猪瘟、猪伪狂犬病、猪流行性腹泻等重要猪病的流行状况,着重总结了非洲猪瘟疫情发生情况。分析了2020年非洲猪瘟等疫病的流行趋势,同时提出了坚持生物安全、严格处置非洲猪瘟疫情等防控策略。     

接种猪瘟兔化弱毒株的永生化猪血管内皮细胞传代情况研究

在传至70代的永生化猪血管内皮细胞接种猪瘟兔化弱毒,72 h后收取细胞上清液并对处理的细胞进行传代培养,分别检测第70、75、80、90、100、105代接毒细胞中病毒,观察各代细胞的生长情况。结果表明,接种猪瘟兔化弱毒后20代(第90代永生化猪血管内皮细胞)的细胞与正常的形态相似,但传至接毒后35代(第105代永生化猪血管内皮细胞)时,细胞稍有拉长现象。各代次细胞中都有猪瘟兔化弱毒的检出。本研究为永生化猪血管内皮细胞生产猪瘟兔化弱毒疫苗奠定了基础。     

Effector mechanisms in the pig. Antibody-dependent cellular cytolysis of African swine fever virus infected cells

Antibody dependent cellular cytolysis (ADCC) against African swine fever virus infected nucleated cells was investigated in a porcine system. Of the peripheral blood components examined, only neutrophils acted as effectors. Lymph node derived cells displayed no ADCC activity. In vitro yield reduction assays suggested that neutrophil mediated ADCC may play a role in recovery from African swine fever virus infection.     

一步法RT-PCR同时检测CSFV和PRRSV方法的建立及应用

猪瘟(CSF)和猪繁殖与呼吸综合征(PRRS)是严重危害云南养猪业的传染病,为了建立一种能快速诊断猪瘟和猪繁殖与呼吸综合征混合感染的方法,根据NCBI上已发表的猪瘟病毒(CSFV)保守基因Erns和猪繁殖与呼吸综合征病毒(PRRSV)保守基因M基因的核苷酸序列,利用Oligo 6.0设计了2对特异性引物,经过PCR反应...     

Serological Diagnosis of Classical Swine Fever: A Comparison of a Modified Direct Complement Fixation Test with an Immunofluorescence Plaque Neutralization Test in the Diagnosis of Experimental Subclinical Infection

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera.In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies.During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.     

Replication of classical swine fever virus strains and isolates in different porcine cell lines

Classical swine fever virus (CSFV) is an economically important pathogen of domestic pigs and wild boar. Due to the highly variable clinical picture of CSF, laboratory methods are essential for an unambiguous diagnosis. Virus isolation using cell culture is still considered the gold standard. It is based on the incubation of permissive cells with organ or leukocyte preparations followed by antigen detection. In the "EU Diagnostic Manual for CSF Diagnosis", the permanent cell line PK(15) (porcine kidney) is recommended. In the European Reference Laboratory (EURL) a clone of this cell line, PK(15)A, and the STE (swine testicular epitheloid) cell line are in use for propagation of CSFV. The aim of this work was to assess the relative ability of eleven permanent cell lines derived from various organs of wild boar and domestic pig, respectively, to support the replication of different strains and isolates in comparison to these cell lines. An avirulent and a highly virulent laboratory CSFV strain, and several recent field isolates from domestic pigs and wild boars were used. Titers were determined after one, two and three virus passages, and after 48 and 120 h of incubation. Of the eleven cell lines analyzed, two were found that replicated all the tested CSFV strains and field isolates. Those may be useful for improving diagnosis of CSFV and for preparing low-passaged virus stocks of new isolates.     

非洲猪瘟病毒p62蛋白单克隆抗体的制备及初步应用

为制备非洲猪瘟病毒（ASFV）p62蛋白的特异性单克隆抗体,并初步应用于感染组织样品中ASFV抗原的免疫组化（IHC）检测,本研究以杆状病毒表达的非洲猪瘟病毒重组p62蛋白免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞进行融合获得杂交瘤细胞。结果显示：基于纯化的p62蛋白建立的间接ELISA方法对杂交瘤细胞进行筛选和亚克隆,获得了18株可稳定分泌抗非洲猪瘟病毒p62蛋白单克隆抗体的杂交瘤细胞株。经IFA检测,制备的单克隆抗体均与非洲猪瘟病毒反应,且不与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型等猪源常见病毒反应,特异性良好。抗体识别蛋白的鉴定结果显示,3株MAbs识别p35蛋白,15株MAbs识别p15蛋白。14株MAbs重链亚类为IgG1型,4株MAbs重链亚类为IgG2a,轻链均为κ链。利用18株MAbs对ASFV感染猪的肺、扁桃体、淋巴结等组织进行IHC检测,结果显示5株MAbs均能够与感染ASFV的组织发生特异性的免疫反应。本研究获得的非洲猪瘟病毒p62蛋白单克隆抗体可为非洲猪瘟病毒免疫学检测方法的建立及p62蛋白的结构功能等基础研究提供重要的生物材料。     

一例猪圆环病毒2型和大肠杆菌、溶血葡萄球菌混合感染的诊断

山东德州某猪场发生猪高热、呼吸系统疾病甚至死亡的疫情。采集病料提取病变组织总DNA或RNA进行猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒、猪瘟病毒的PCR或RT-PCR检测。PCR扩增出353 bp的猪圆环病毒2型特异性条带。同时进行细菌分离培养、生化鉴定等试验,诊断为猪圆环病毒2型和大肠杆菌、溶血葡萄球菌混合感染。     

PPARγ对猪血管内皮细胞增殖、迁移及小管形成的影响

试验旨在研究过氧化物酶体增殖物激活受体γ（peroxisome proliferator-activated receptors γ,PPARγ）对猪血管内皮细胞增殖、迁移及小管形成的影响,并探讨PPARγ在猪体外血管生成中的作用。设置PPARγ激动剂组（5、10、15、20 μmol/L罗格列酮）、抑制剂组（5、10、15、20 μmol/L T0070907）及对照组,通过iCelligence细胞功能分析、划痕试验和Matrigel基质胶三维培养,构建猪体外血管生成的模型,模拟猪体内血管生成的环境,分别对猪血管内皮细胞的增殖、迁移、小管形成能力进行测定,同时根据NCBI已有的相关序列,应用Primer Premier 5.0软件设计PPARγ基因特异性引物,利用SYBR GreenⅠ实时荧光定量PCR检测PPARγ基因mRNA相对表达量,对PPARγ的体外作用效果进行验证。结果显示,5、10 μmol/L罗格列酮能促进猪血管内皮细胞增殖、迁移及小管形成,T0070907的抑制效果在试验浓度区间内（5~15 μmol/L）随浓度升高而加强,较高浓度（20 μmol/L）的两种药物均由于药物毒性的影响对细胞活动产生干扰。此外,5~20 μmol/L罗格列酮和5~20 μmol/L T0070907能分别提高和降低PPARγ基因mRNA相对表达量,且浓度趋势与增殖、迁移、小管形成的试验结果一致。综上所述,通过激活PPARγ可以对猪血管内皮细胞的增殖、迁移及小管形成产生促进效果,提示其在猪体外血管生成中具有积极作用,可为研究PPARγ对猪胎盘血管发生的影响提供参考依据。     

Cell type-specific resistance of trigeminal ganglion neurons towards apoptotic stimuli

Trigeminal ganglion (TG) neurons are important target cells for many alphaherpesviruses and constitute a major site of virus latency and reactivation. Earlier we showed that porcine TG neurons are remarkably more resistant towards (apoptotic) cell death resulting from infection by the swine alphaherpesvirus pseudorabies virus (PRV) compared to a broad range of other primary porcine cell types and that this resistance does not depend on the strongly anti-apoptotic US3 viral protein kinase (Geenen, K., Favoreel, H.W., Nauwynck, H.J., 2005a. Higher resistance of porcine trigeminal ganglion neurons towards pseudorabies virus-induced cell death compared with other porcine cell types in vitro. J. Gen. Virol. 86, 1251-1260). Although other viral anti-apoptotic proteins may be involved in survival of TG neurons during PRV infection, an additional factor may be that TG neurons possess a cell type-dependent capacity to withstand apoptosis compared to other cell types. To investigate this, we treated uninfected porcine TG cultures, swine kidney cells, and porcine superior cervical ganglion (SCG) neurons with several apoptosis-inducing reagents (staurosporine, camptothecin and genistein). None of these reagents were able to trigger substantial apoptotic cell death in TG neurons, whereas non-neuronal TG cells, swine kidney cells, and SCG neurons showed a clear dose-dependent increase in apoptosis using either of these reagents. In conclusion, sensory TG neurons may contain a cell type-specific capacity to withstand different apoptotic assaults, including infection with an alphaherpesvirus.     

IL-13 replaces IL-4 in development of monocyte derived dendritic cells (MoDC) of swine

Dendritic cells (DCs) are a critical aspect of innate immune responses in addition to initiating adaptive immunity. In vitro generation of monocyte derived dendritic cells (MoDC) by culturing cells in IL-4 and granulocyte/macrophage colony stimulating factor (GM-CSF) has been reported for multiple species including swine. However, IL-4 is not a prominent cytokine detected in the periphery of common breeds of swine such as Yorkshire pigs. In this study, we report the generation and characterization of porcine MoDC in vitro using porcine IL-13 and porcine GM-CSF. These cells have the predicted expression of Class II MHC and T cell costimulatory molecules, phagocytic capacity and the ability to process and present antigen. Critically, porcine IL-13/GM-CSF MoDC have the unique ability to stimulate a primary mixed lymphocyte response in vitro. The type I interferon response of these MoDC to poly I:C (TLR3 ligand), LPS (TLR4 ligand) and CpG (TLR9 ligand) was tested. Of these TLR agonists, LPS or CpG did not stimulate induction of type I interferons, but a strong response was observed to poly I:C. This analysis shows that the generation of MoDCs in IL-13 yields cells of equivalent phenotype and function as IL-4 generated DC. However, for swine, in vitro generation of MoDC in IL-13 is likely to induce a more physiological cell population to study given expression of IL-4 is lacking in the periphery of these animals.     

猪伪狂犬病毒PCR检测方法的建立及应用

根据猪伪狂犬病毒（PRV）gE基因序列保守区段,设计一对特异性引物,通过优化反应条件,建立了可区分猪伪狂犬病毒野毒株与基因缺失疫苗株的PCR检测方法,并对该方法的敏感性、特异性和重复性进行了验证。结果显示,该PCR方法可扩增出388 bp的目的片段;对模板的最低检测量为1.1 pg;与猪圆环病毒Ⅱ型、猪细小病毒、猪支原体、猪瘟病毒、猪繁殖与呼吸综合征病毒、猪乙型脑炎病毒、猪流行性腹泻病毒无交叉反应,具有高特异性。采用建立的PCR方法对2014年以来全国不同地区81个猪场421份疑似病料进行检测,发现PRV猪场平均阳性率为35.80%,样品平均阳性率为 25.42%。该方法灵敏度高、特异性强、重复性好,可用于PRV的临床诊断和流行病学调查。