Molecular Evidence Of Transplacental (Vertical) Route Of Transmission Of African Swine Fever In Foetus Of Pig: A Case Report

J.F. Antiabong; O. A. Owolodun; O. A. Adefalujo; B. Yakubu; M. E. Ogedengbe; D. Shamaki

摘要

African Swine Fever (ASFV) DNA was detected in the Placenta, and organs of fetuses of a sacrificed sow using the polymerase chain reaction technique.These findings suggest a transplacental (vertical) route of transmission of ASFV in the pathogenesis of African swine fever in foetus of pig and calls for attention considering its epizootiological potential. Introduction African swine fever is the only member of the newly named Asfarviridae whose genomic organization and cytoplasmic replication is similar to that of poxviridae (Salas, 1999). The genomic size of ASFV varies between 170kb and 190kb with variable regions which contains at least five multigene families (vinuela , 1985;costa, 1990).The disease was first detected in Kenya in 1910 (Montgomery, 1921) and occurred outside Africa for the first time in Lisbon (Portugal) in 1957 (Manso Ribeivo et al, 1985). The first outbreak in Nigeria occurred in Lagos between 1997 and early 1998 and was isolated by odemuyiwa et al, 1999.As the only known DNA arbovirus it cycles between ornithodorous ticks and wild pig populations (Warhogs and Bush Pigs) in Sub-Saharan Africa (Wilkinson, 1984). ASFV can be transmitted by contact as the virus is present in sufficient amounts in secretions and extractions. The virus is very stable in the blood and faeces of infected animals. Airborne transmission over short distances can also occur (Salas, 1999).The placenta is the obvious route for passage of infectious agent from mother to the offspring and for this to occur the virus must be present in the blood of the animal at the right stage of gestation and must reach the placenta and then the foetus. Transplacental infection has been reported for animal diseases such as hog cholera, porcine parvovirus, mucosal disease and equine infectious anaemia (Mims, 1981).In an experimental infection of pregnant sows ,ASF virus was recovered from foetal placenta, amniotic fluid and heart blood with specific immunoflorescence present only in the placenta tissues (Schlafer and Mebus ,1987) suggesting the possibility of transplacental infection of the foetus. In this study we show a PCR evidence of a natural transplacental infection of the foetus by the African swine fever virus. Materials And Methods Source of SamplesTissue samples were collected aseptically from internal organs (liver, heart, kidney, lungs) and placenta of fetuses removed from a sacrificed pregnant sow that belonged to a piggery that recorded mortality of pigs of all ages, recumbence, anorexia, varying degrees of diarrhea, reddening of skin of affected pigs. Liver tissue was collected from the sow.Tissue Homogenization0.5g of each tissue sample was homogenized using Ultra Turrax T8 (Ika- werke?) 2mls of phosphate buffer saline (PBS) was added to each of the homogenate and then short spinned at 12,000rpm for 20 seconds in microcentifuge tubes (Eppendorf?) to obtain the supernatant which contains the virus.Genomic DNA ExtractionThe genomic DNA was extracted from 100μl of the supernatants using the Tripure reagent (Roche?) according to the manufacturer's instruction. Extracted DNA was then transferred into a clean, nuclease free microcentrifuge tubes and stored at -200C for further analysis.Master mix/AmplificatioA diagnostic PCR was performed using 3.0μl template DNA extract in a total reaction mix of 25μl. The reaction mix included 14.5μl at nuclease free water (Promega ?), 4.5μl Dynazyme buffer (1x10mM Tris-HCl pH8.8, 1.5mM MgCl2,50mM Kcl, 0.1% Triton x – 100) Promega ?), 0.5μl dNTP (10mM nucleotide mix) (Promega?), 20pMol of primers PAS1 ( sense) S1 – ATG GAT ACC GAG GGA ATA GC-31 and PAS2 (anti sense) S1 – CTT ACC GAT GAA AAT GAT AC – 31 (GIBCO brl?) which amplifies a 278bp fragment of the VP72 gene of ASFV genome, 0.5μl of Tag.DNA polymerase (Roche?). 20μl of mineral oil (Sigma?) was added to prevent evaporation. Amplification of target DNA sequence was carried out in a cyclogene thermal cycler (Techne?) at initial denaturation of 940C for 15s

机译：使用聚合酶链反应技术在胎盘和处死的母猪的胎儿器官中检测到非洲猪瘟（ASFV）DNA，这些发现表明ASFV的胎盘（垂直）传播途径在胎儿非洲猪热的发病机理中猪，鉴于其流行病学潜力，需要引起注意。简介非洲猪瘟是新命名的Asfarviridae的唯一成员，其基因组组织和细胞质复制与poxviridae相似（Salas，1999）。 ASFV的基因组大小在170kb和190kb之间变化，具有可变区，至少包含五个多基因家族（vinuela，1985; costa，1990）。该病于1910年在肯尼亚首次发现（蒙哥马利，1921年），并在非洲以外的地方发生。 1957年第一次在里斯本（葡萄牙）（Manso Ribeivo等，1985）。尼日利亚的第一次暴发发生在1997年至1998年初的拉各斯，并由odemuyiwa等人于1999年分离出。作为唯一已知的DNA虫媒病毒，它在撒哈拉以南非洲的虎口tick和野猪种群（Warhogs和Bush Pigs）之间循环（威尔金森（1984）。由于病毒以足够的量存在于分泌物和提取物中，因此可以通过接触传播ASFV。该病毒在被感染动物的血液和粪便中非常稳定。空中传播也可能发生在短距离内（Salas，1999）。胎盘是传染病从母体传给后代的明显途径，为此，病毒必须在正确的阶段出现在动物的血液中妊娠，必须到达胎盘，然后到达胎儿。已有报道称经胎盘感染可引起猪霍乱，猪细小病毒，粘膜病和马传染性贫血等动物疾病（Mims，1981）。在实验性怀孕母猪感染中，从胎盘，羊水和心脏血液中回收了ASF病毒。仅在胎盘组织中存在特异性免疫荧光（Schlafer和Mebus，1987），提示经胎盘感染胎儿的可能性。在这项研究中，我们显示了非洲猪瘟病毒对胎儿自然经胎盘感染的PCR证据。材料和方法样品来源组织样品是从内脏器官（肝脏，心脏，肾脏，肺脏）和胎盘中无菌采集的，这些胎儿是从处死的母猪身上摘下来的，这些母猪属于猪场，记录了各个年龄段的猪的死亡率，卧倒，厌食，不同程度的腹泻，患病猪的皮肤变红。从母猪中收集肝组织。组织匀浆使用Ultra Turrax T8（Ikawerke？）匀浆0.5g每个组织样品，向匀浆中加入2ml磷酸盐缓冲盐水（PBS），然后以12,000rpm的速度短时间离心在微量离心管（Eppendorf？）中放置20秒钟，以获得含有病毒的上清液。基因组DNA提取根据制造商的说明，使用Tripure试剂（Roche？）从100μl上清液中提取基因组DNA。然后将提取的DNA转移到干净的无核酸酶的微量离心管中，并保存在-200°C进行进一步分析。预混液/扩增使用3.0μl模板DNA提取物在25μl的总反应混合物中进行诊断性PCR。反应混合物包括在无核酸酶的水（Promega？）上的14.5μl，4.5μl的Dynazyme缓冲液（1x10mM Tris-HCl pH8.8、1.5mM MgCl2、50mM Kcl，0.1％Triton x – 100）Promega？），0.5μldNTP（ 10mM核苷酸混合物）（Promega？），20pMol引物PAS1（正义）S1 – ATG GAT ACC GAG GGA ATA GC-31和PAS2（反义）S1 – CTT ACC GAT GAA AAT GAT AC – 31（GIBCO brl？）其中扩增了ASFV基因组VP72基因的278bp片段，即0.5μlTag.DNA聚合酶（Roche？）。加入20μl矿物油（Sigma TM）以防止蒸发。在940℃的初始变性条件下，在回旋热循环仪（Techne？）中进行靶DNA序列的扩增。

Molecular Evidence Of Transplacental (Vertical) Route Of Transmission Of African Swine Fever In Foetus Of Pig: A Case Report

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