The lesional changes and pathogenesis in the kidney in african swine fever

African swine fever is a viral haemorrhagic disease of pigs which has been used as a model for the study of viral haemorrhagic diseases in man. The acute course of the disease is characterized by acute proliferative glomerulonephritis, with viral replication in mesangial cells and occasional focal necrosis of the renal tubular system; hyperplasia of the collecting ducts is associated with evident virus replication. Haemorrhages have been attributed to endothelial dysfunction, aggravated by virus replication in endothelial cells in the final stages of the disease. The renal interstitium displays intense oedema and an infiltrate largely composed of macrophages. Virus replication has also been observed in fibroblasts and in the smooth0muscle cells of arterioles and venules.In subacute-chronic forms of the disease, various types of glomerulonephritis are observed, ranging from mesangial proliferative glomerulonephritis to focal and segmental hyalinosis associated with immune-mediated phenomena. No striking changes are reported in the renal tubular system. Interstitial haemorrhages are associated with diapedesis due to immunologically mediated events. The interstitium has also been found to contain a lymphohistiocytic infiltrate with abundant plasma cells. No evidence has been reported of viral replication in any cell population.Abbreviations ASF African swine fever - ASFV African swine fever virus - C complement component - DNA deoxyribonucleic acid - HAD₅₀ 50% haemoadsorbing doses - Ig immunoglobulin - MPS mononuclear phagocytic system - PAS periodic acid-Schiff reagent     

急性非洲猪瘟脑损伤及NF-κB介导脑水肿形成机制研究

【目的】 在试验条件下研究非洲猪瘟病毒（African swine fever virus，ASFV）所致急性脑损伤及核因子κB （NF-κB）介导脑水肿发生的相关病理机制。【方法】 18头健康长白猪随机分为2组，攻毒组（13头）和对照组（5头），攻毒组肌内注射剂量为10²HAD₅₀/mL （HAD₅₀：半数红细胞吸附量）的ASFV毒株Pig/HLJ/18，对照组注射等体积生理盐水，试验期为15 d。试验期间观察临床症状，剖检死亡猪并观察脑部病变；利用原位PCR检测进行病毒定位；HE染色及甲苯胺蓝染色观察脑组织病理变化；免疫组化染色法检测脑组织中肿瘤坏死因子-α（TNF-α）、白介素-1β（IL-1β）、干扰素-γ（IFN-γ）、NF-κB、基质金属蛋白酶9（MMP-9）、水通道蛋白4（AQP-4）的表达。【结果】 攻毒组9头猪表现出临床神经症状，脑部剖检病变主要为脑膜充血和不同程度的脑实质水肿；ASFV主要位于脑部微血管中；病变早期主要为脑水肿，脑皮质区结构疏松、血管周围间隙增宽、神经元肿胀变圆、染色变淡，中后期除了表现脑水肿的特征外，还可见脑血管充血、多量微血栓形成及淋巴细胞浸润；免疫组化结果显示，攻毒组TNF-α、IL-1β、IFN-γ、NF-κB及MMP-9主要在神经元及胶质细胞中表达，AQP-4的阳性细胞主要是微血管周围的星形胶质细胞，与对照组相比，攻毒组上述6种因子的阳性表达率均极显著增高（P<0.01）。【结论】 急性非洲猪瘟脑损伤的主要表现为脑膜血管充血出血和脑实质水肿、组织学病变显示病毒性脑炎，ASFV感染脑部，促进炎性因子的异常高表达，通过激活NF-κB信号通路对TNF-α、IL-1β、IFN-γ的转录进行调控，使这几种炎性因子的产生和释放增多，扩大炎症反应，影响血脑屏障的通透性，还能上调MMP-9与AQP-4的表达，破坏血脑屏障，促进脑水肿的发生发展。     

Experimental Pathological Study of Acute African Swine Fever

To study the clinicopathology and histopathology of African swine fever (ASF), and to explore the internal relationship between pathological changes and disease occurrence and development and its pathological mechanism, 13 Landrace pigs with bodyweight about 20 kg were intramuscular injected with African swine fever virus (ASFV), strain Pig/HLJ/18 at a dose of 10²HAD₅₀·mL^-1. During the experiment, all the dead pigs were systematically dissected and sampled, paraffin sections were produced, and haematoxylin-eosin staining was performed. Clinicopathological evaluation standards for acute ASF were established, then pathological lesions (classification variables) were expressed by counting frequency and percentage, and the lesion degree (continuous variables) was graded and scored according to different pathological changes of various tissues and organs. The results showed that all infected pigs were in line with the clinical characteristics of ASF, including acute, febrile and highly infectious, with a 100% incidence rate and 100% mortality. The dead pigs showed typical characteristics of septicemia, cadavers prone to corruption, blood clotting adverse or hemolysis, rigor mortis incomplete. The main pathological lesions were hemorrhagic necrotizing lymphadenitis, acute inflammatory splenomegaly (septic spleen), cerebral edema, pulmonary edema and lung consolidation et al. The spleen and lymphonodus are the target organs attacked by ASFV, with the most significant lesions, the earliest occurrence time, the longest duration and the highest frequency. The most prominent pathological changes are blood circulation disorders, including multiple pathological manifestations such as edema, hyperemia, congestion, hemorrhage, infarction, disseminated intravascular coagulation (DIC), and the most important characteristics are hemorrhagic lesions. The inflammatory reaction of lymphocytic exudation caused by ASFV runs through the whole process, especially in the middle and later stages of the course. The results suggest that the main pathological process of acute African swine fever is a typical immune/inflammatory cascade reaction and severe systemic blood circulation disorder, which resulted in the high incidence rate and high mortality rate of acute ASF.     

急性非洲猪瘟的实验病理学研究

旨在通过对非洲猪瘟临床病理学和组织病理学的研究,探讨病理学变化与疾病发生发展的内在关系及其病理机制。选用体重20 kg左右的长白猪13头,肌内注射非洲猪瘟病毒毒株Pig/HLJ/18,剂量10²HAD₅₀·mL^-1。试验期间的死亡猪,全部进行系统剖检和取材,制备石蜡切片,苏木素伊红染色。建立病理学评价标准,病变（无序分类变量）用频率和百分比表示,病变程度（有序分类变量）按各组织器官的不同病变进行分级和评分。结果表明,发病猪符合非洲猪瘟急性、热性、高传染性等临床特征,发病率100%,病死率100%。病死猪表现败血症典型特征,尸体易腐败,血凝不良或溶血,尸僵不全。主要病理损伤为出血性坏死性淋巴结炎、急性炎性脾肿（败血脾）、脑水肿、肺水肿和肺实变等。脾和淋巴结是非洲猪瘟病毒攻击的靶器官,病变最为显著,出现时间最早,持续时间最长,发生频率最高。病理变化以血液循环障碍尤为突出,包括水肿、充血、淤血、出血、梗死和弥散性血管内凝血等多种病理表现,出血性病变为其最主要的特征。非洲猪瘟病毒引发的以淋巴细胞渗出为主的炎症反应贯穿始终,在病程的中后期表现更为明显。结果提示,急性非洲猪瘟的主要病理过程为典型的免疫/炎症级联反应和严重的全身血液循环障碍,共同导致急性非洲猪瘟的高发病率和高死亡率。     

非洲猪瘟病原学及单克隆抗体制备技术研究进展

非洲猪瘟是由非洲猪瘟病毒引起猪的高度接触性、传染性、出血性以及高死亡率的传染病。20世纪中期以来,已在非洲、欧洲和美洲等数十个国家流行,并在近几年内蔓延至欧亚两洲接壤处的格鲁吉亚、亚美尼亚、阿塞拜疆以及俄罗斯境内,其一旦侵入我国,将会给我国养猪业带来极大的危害。非洲猪瘟病原学研究以及制备相应的单克隆抗体对非洲猪瘟病毒快速诊断技术研究和疫苗研制有着重大的现实意义。主要从病原学和单克隆抗体制备方面对非洲猪瘟的研究进展进行了综述。     

非洲猪瘟疫苗研究进展

非洲猪瘟（African swine fever,ASF）是由非洲猪瘟病毒（African swine fever virus,ASFV）引起的家猪和野猪的急性、出血性传染病,强毒株感染猪的致死率接近100%,给养猪业造成巨大经济损失。ASFV在猪群中可以快速有效传播,在环境中可稳定存在,为ASF的防控带来了挑战。研究人员一直致力于ASF疫苗的研究,迄今为止,仍没有有效的疫苗投入市场。综述了ASF灭活疫苗、减毒活疫苗、亚单位疫苗、DNA疫苗、病毒活载体疫苗的研究情况,以期为ASF疫苗的有效研发提供参考。     

鉴别非洲猪瘟病毒和猪瘟病毒野毒株二重TaqMan MGB实时荧光定量PCR检测方法的建立及初步应用

旨在建立特异、敏感的实时荧光定量PCR（FQ-PCR）方法,用于非洲猪瘟病毒（ASFV）和猪瘟病毒（CSFV）野毒株的快速鉴别检测。针对ASFV的P72基因和CSFV野毒株的5'UTR非编码区序列的保守区域分别设计1对特异性引物和1条探针,经优化反应条件,建立一种基于TaqMan MGB探针技术的FQ-PCR方法,验证方法的敏感性、特异性和稳定性,对50份临床样品进行检测,并与猪瘟国标方法及OIE推荐的非洲猪瘟检测方法进行比较分析。结果显示：建立的鉴别ASFV和CSFV野毒株二重FQ-PCR检测方法在10⁰~10⁶拷贝·μL^-1模板范围内有良好的线性关系;对ASFV和CSFV基因出现阳性扩增信号,但对猪瘟病毒疫苗株、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、猪细小病毒、猪乙型脑炎病毒、副猪嗜血杆菌等病原对照未出现扩增;批内、批间试验变异系数在1.18%~2.08%,重复性良好;对ASFV和CSFV的最低检测模板浓度均为10拷贝·μL^-1;利用建立的二重FQ-PCR方法对50份临床样品进行检测,检测结果与猪瘟国标方法及OIE推荐的非洲猪瘟检测方法结果完全一致。本研究成功建立了鉴别ASFV和CSFV野毒株二重TaqMan MGB FQ-PCR方法,为ASFV和CSFV野毒株的鉴别诊断提供了快速、敏感、特异且能满足临床检测需求的检测方法。     

Risk factors of African swine fever virus in suspected infected pigs in smallholder farming systems in South-Kivu province,Democratic Republic of Congo

BackgroundAfrican swine fever (ASF) is an infectious viral disease of domestic pigs that presents as a hemorrhagic fever, and for which no effective vaccine is available. The disease has a serious negative social and economic impact on pig keepers. There is limited information on the potential risk factors responsible for the spread of ASF in South Kivu.ObjectiveThe aim of this study was to determine the potential risk factors associated with ASF infection in suspected ASF virus (ASFV)-infected pigs.MethodsWe sampled whole blood from 391 pigs. Additionally, 300 pig farmers were interviewed using a structured questionnaire. Viral DNA was detected by using the real-time polymerase chain reaction technique.ResultsThe majority of pigs sampled, 78% (95% confidence interval [CI], 74.4–82.6), were of local breeds. Over half, 60.4% (95% CI, 55.5–65.2), were female, and most of them, 90.5% (95% CI, 87.6–93.4), were adult pigs (> 1 year old). Viral DNA was detected in 72 of the 391 sampled pigs, indicating an overall infection rate of 18.4% (95% CI, 14.5–22.4). Multivariable logistic regression analysis revealed several risk factors positively associated with ASFV infection: feeding with swill in pen (odds ratio [OR], 3.8; 95% CI, 2.12–6.77); mixed ages of pigs in the same pen (OR, 3.3; 95% CI, 1.99–5.57); introduction of new animals to the farm (OR, 5.4; 95% CI, 1.91–15.28). The risk factors that were negatively (protective) correlated with ASFV positivity were the presence of male animals and the use of an in-pen breeding system.ConclusionLocal pig farmers should be encouraged to adopt proper husbandry and feeding practices in order to increase the number of ASF-free farms.     

Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response

Classical swine fever (CSF) is an economically important swine disease worldwide. The glycoprotein E2 of classical swine fever virus (CSFV) is a viral antigen that can induce a protective immune response against CSF. A recombinant E2 protein was constructed using the yeast Pichia pastoris expression system and evaluated for its vaccine efficacy. The yeast-expressed E2 (yE2) was shown to have N-linked glycosylation and to form homodimer molecules. Four 6-week-old specified-pathogen-free (SPF) piglets were intramuscularly immunized with yE2 twice at 3-week intervals. All yE2-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:96 to 1:768. Neutralizing antibody titers at 10 weeks post booster vaccination ranged from 1:16 to 1:64. At this time, the pigs were subjected to challenge infection with a dose of 1 × 10⁵ TCID₅₀ (50% tissue culture infective dose) virulent CSFV strain. At 1 week post challenge infection, all of the yE2-immunized pigs were alive and without symptoms or signs of CSF. Neutralizing antibody titers at this time ranged from 1:4,800 to 1:12,800 and even to 1:51,200 one week later. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 6 days post challenge infection. All of the yE2-vaccinated pigs were E^rns antibody negative and had seroconverted against E^rns by post challenge day 11, suggesting that yE2 is a potential DIVA (differentiating infected from vaccinated animals) vaccine. The yeast-expressed E2 protein retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.     

Development of a ladder-shape melting temperature isothermal amplification (LMTIA) assay for detection of African swine fever virus (ASFV)

BackgroundDue to the unavailability of an effective vaccine or antiviral drug against the African swine fever virus (ASFV), rapid diagnosis methods are needed to prevent highly contagious African swine fever.ObjectivesThe objective of this study was to establish the ladder-shape melting temperature isothermal amplification (LMTIA) assay for the detection of ASFV.MethodsLMTIA primers were designed with the p72 gene of ASFV as the target, and plasmid pUC57 was used to clone the gene. The LMTIA reaction system was optimized with the plasmid as the positive control, and the performance of the LMTIA assay was compared with that of the commercial real-time polymerase chain reaction (PCR) kit in terms of sensitivity and detection rate using 200 serum samples.ResultsOur results showed that the LMTIA assay could detect the 10⁴ dilution of DNA extracted from the positive reference serum sample, which was the same as that of the commercial real-time PCR kit. The coincidence rate between the two assays was 100%.ConclusionsThe LMTIA assay had high sensitivity, good detection, and simple operation. Thus, it is suitable for facilitating preliminary and cost-effective surveillance for the prevention and control of ASFV.     

Comparative susceptibility of indigenous and improved pig breeds to Classical swine fever virus infection: Practical and epidemiological implications in a subsistence-based, developing country setting

This study investigated the comparative susceptibility of indigenous Moo Laat and improved Large White/Landrace pig breeds to infection with classical swine fever virus (CSFV) under controlled conditions in the Lao People’s Democratic Republic (Lao PDR). The Moo Laat (ML) and Large White/Landrace crossbreed (LWC) pigs were inoculated with a standard challenge strain designated Lao/Kham225 (infectivity titre of 10^2.75 TCID₅₀/ml). The results demonstrated that both the native breed and an improved pig breed are fully susceptible to CSFV infection and the mortality rate is high. LWC pigs demonstrated lower (or shorter) survival times (50% survival time: 11 days), earlier and higher pyrexia and earlier onset of viraemia compared to ML pigs (50% survival time: 18 days). In the context of village-based pig production, the longer time from infection to death in native ML pigs means that incubating or early sick pigs are likely to be sold once an outbreak of CSF is recognized in a village. This increased longevity probably contributes to the maintenance and spread of disease in a population where generally the contact rate is low.     

非洲猪瘟病毒传播方式及控制措施

非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fever virus,ASFV)引起的一种急性、热性、高度传染性疫病,感染猪以发热和全身性出血为主要特征,病程短、病死率高。非洲猪瘟主要流行于非洲国家,随后相继传入西欧、南美洲、东欧,以及亚洲国家,对全球养猪业、食品安全和猪及其产品国际贸易产生了严重危害和深远影响。现结合参考文献和我国非洲猪瘟发生、控制情况,分析了非洲猪瘟病毒传播动力学、传播方式,提出防控非洲猪瘟的主要措施。     

1株非洲猪瘟病毒自然变异毒株的鉴定

非洲猪瘟(ASF)于2018年传入我国后已流行了2年有余,我国非洲猪瘟病毒(ASFV)毒株未曾出现较大变异.最近,针对我国ASF疫情放缓以及感染猪出现的低死亡率现象,我们在ASFV生态学研究中,从主动监测的样品中分离到1株源自湖北某地的ASFV自然变异株.经基因组测序发现,其EP402R基因(CD2v)和上游相邻的EP...     

Updated distribution and host records for the argasid tick Ornithodoros (Pavlovskyella) zumpti: A potential vector of African swine fever virus in South Africa

African swine fever virus (ASFV) causes a lethal and contagious disease of domestic pigs. In South Africa, the virus historically circulated in warthogs and ornithodorid ticks that were only found in warthog burrows in the north of the country. Regulations implemented in 1935 to prevent transfer of infected animals or products to the south initially proved effective but from 2016 there have been outbreaks of disease in the south that cannot be traced to transfer of infection from the north. From 1963 there were widespread translocations of warthogs to the south, initially from a source considered to be free of ornithodorid ticks. We undertook to determine whether sylvatic circulation of ASFV occurs in the south, including identification of potential new vectors, through testing extralimital warthogs for antibody and ticks for virus. Results of testing warthogs for antibody and other species of ticks for virus will be presented separately. Here we report finding Ornithodoros (Pavlovskyella) zumpti ticks in warthog burrows for the first time. This occurred in the Eastern Cape Province (ECP) in 2019. Since African swine fever was recognised in the ECP for the first time in 2020 and outbreaks of the disease in domestic pigs continue to occur there, priority should be given to determining the distribution range and vector potential of O. (P.) zumpti for ASFV.     

Comparison of antibody values in sera of pigs vaccinated with a subunit or an attenuated vaccine against classical swine fever

Ten pigs, aged 85 days, were vaccinated with a subunit vaccine containing 32 g of classical swine fever virus glycoprotein E2 (gp E2) (group 1), and a further 10 pigs were vaccinated with a C strain vaccine (10^4±0.15 TCID₅₀/ml), produced by amplification in minipig kidney (MPK) cell culture (group 2). Nine non-vaccinated pigs served as a control group (group 3). Serum samples were collected before (day 0) and at 4, 10, 21 and 28 days after vaccination and were analysed by two commercially available enzyme immunoassays and by a neutralizing peroxidase-linked assay (NPLA). At the same times, peripheral blood was taken for determining the total leukocyte count and the body temperature was taken daily. Antibodies were not detected in serum samples collected before vaccination (day 0), and no side-effects that could be connected with vaccination were observed during the trial. Ten days after vaccination 6/10 pigs vaccinated with the subunit vaccine were seropositive. On days 21 and 28, the ratios of serologically positive to vaccinated pigs were 9/10 and 10/10, respectively. Four of the ten pigs that were vaccinated with the C strain vaccine were positive on day 21 and 9/10 on day 28. However, the results of the NPLA showed that only 4/10 pigs had an antibody titre >1:32 at the end of the trial in both the vaccinated groups, even though the subunit vaccine initiated an earlier and higher level of neutralizing antibodies than the vaccine produced from the C strain. Challenge was performed 28 days after vaccination on four randomly selected pigs from both vaccinated groups. The pigs survived the challenge without showing any clinical signs of classical swine fever (CSF), while two nonvaccinated control pigs died on the 10th and 12th days after infection.     

吸血性节肢动物在非洲猪瘟病毒传播过程中可能扮演的角色

非洲猪瘟自2018年冬季从国外传入,给国内的生猪产业造成毁灭性打击,已造成大量生猪死亡和众多养殖户恐慌.尽管非洲猪瘟已被世界动物卫生组织列为法定上报疾病之一,但是市面上尚无有效的治疗药物和疫苗,故国际上针对该病的防制措施仍停留于消灭传染源、切断传播途等物理防御.据文献记载,非洲猪瘟病毒可由某些吸血性节肢动物所传播,故清...     

用间接免疫荧光检测方法评价猪瘟兔化弱毒活疫苗效力的研究

为用体外试验方法评价猪瘟兔化弱毒活疫苗效力,以猪瘟病毒抗体（兔源）为一抗、荧光素标记羊抗兔IgG 为二抗,建立了CSFV兔化弱毒株的间接免疫荧光检测方法（ IFA）。特异性试验表明,用该IFA方法检测CSFV兔化弱毒株接种的RK细胞为阳性,而检测伪狂犬病毒、猪细小病毒病、牛病毒性腹泻/粘膜病毒接种的RK细胞均为阴性。 CSFV兔化弱毒株和活疫苗的兔体感染量（ RID）用兔体测定,半数组织感染量（ TCID50）用IFA测定,拟合RID与TCID50的线性回归方程,确定1RID/mL＝（ TCID50/0．1mL＋200）/12。     

Ecological studies of Yersinia enterocolitica. II. Experimental infection with Y. enterocolitica in pigs

Fattening pigs were inoculated with the human pathogenic strains of Yersinia enterocolitica, biovar 4 serovar 3 phagovar 8 and biovar 2 serovar 5.27. Each pig received 2.6 × 10⁹ organisms by catheter into the stomach and thereafter 10% sodium bicarbonate solution, by the same route. The serovar 3 strain became established in the intestines of four out of six 11-week-old pigs and in all three 24-week-old pigs. Serovar 5.27 established in the intestines of all four 10-week-old pigs. In these pigs, both serovars were excreted in the feces at 10²?10⁶ cells g^?1 for a few weeks. However, the serovar 5.27 was excreted in the feces at less than 10^3·3 cells g^?1 for 2 weeks by 24-week-old pigs. Horizontal transmission with the serovar 5.27 strain was observed on the 5th–11th day, and the bacteria were present in the intestines during the 2nd week. However, the serum titers to O-agglutinin were 1/10 and less, but in one pig, the titer was 1/40 against serovar 5.27 strain.     

非洲猪瘟病毒诱导猪红细胞凋亡并促进猪外周血单核细胞的吞噬作用

本研究旨在探讨非洲猪瘟病毒（ASFV）对猪红细胞（RBCs）的作用以及对猪外周血单核细胞（PBMs）吞噬能力的影响。试验采用流式细胞术检测ASFV侵染猪原代肺泡巨噬细胞（PAMs）和猪红细胞（RBCs）的能力,并检测ASFV诱导RBCs发生凋亡的百分比;同时采用激光共聚焦试验（Confocal）观察ASFV诱导RBCs发生凋亡是否影响PBMs的吞噬能力。结果显示,ASFV不能入侵RBCs,但以时间和剂量依赖性方式诱导RBCs发生凋亡。0.1 MOI ASFV接种RBCs后1、3、5和7 d可分别诱导1.27%、3.23%、7.39%和8.56%的RBCs发生凋亡;1 MOI ASFV接种RBCs后1、3、5和7 d可分别诱导1.54%、3.73%、8.46%和10.74%的RBCs发生凋亡;3 MOI ASFV接种RBCs后1、3、5和7 d可分别诱导2.65%、5.01%、12.44%和18.61%的RBCs发生凋亡。同时,凋亡的RBCs可以增加PBMs对黄绿色荧光微球的吞噬数量,ASFV诱导RBCs凋亡的百分比越高,PBMs吞噬黄绿色荧光微球的数量越多。综上所述,ASFV不能侵染猪RBCs,但可以以时间和剂量依赖性方式诱导RBCs发生凋亡并增强PBMs的吞噬功能。