养殖克氏原螯虾体内白斑综合征病毒的绝对定量分析

近年来克氏原螯虾的养殖受到WSSV的威胁,病毒在宿主组织中的绝对定量对于了解病毒的致病性具有重要意义,但克氏原螯虾组织中WSSV的绝对定量分布还有待研究。实验调查了湖北省5个主养区克氏原螯虾WSSV的感染率,结果表明80%以上克氏原螯虾都携带有WSSV。采用WSSV-VP28蛋白特异性抗体对克氏原螯虾提取蛋白进行Western Blot检测,在WSSV-PCR阳性样品中可检测到VP28特异性条带,在WSSV-PCR阴性样品中没有检测到相应条带。采用实验室建立的WSSV绝对定量PCR方法,对携带病毒的克氏原螯虾6个组织(鳃、胃、肠、血淋巴细胞、肝胰腺和心脏)进行检测。结果表明,在鳃、胃和肠可检测到较多病毒量(约108拷贝/mg),其次是血淋巴细胞(107拷贝/mg)、肝胰腺(106拷贝/mg),在心脏中病毒的含量最低(103拷贝/mg),表明病毒的复制存在组织特异性。结果显示WSSV主要存在于消化系统中,预示着克氏原螯虾可能主要在摄食过程中感染WSSV;不同地区克氏原螯虾组织病毒携带量表现出一定差异,预示着WSSV感染可能受到环境因素的影响。

Quantitative analysis of white spot syndrome virus in the tissues of cultured red swamp crayfish (Procambarus clarkii)

In the late 1930s, red swamp crayfish (Procambarus clarkii ) was introduced into China. Thereafter, it has established its own population in the Yangtze river area and has become an important resource of fresh water prawns in China. In recent years, the amount of wild-caught red swamp crayfish has declined rapidly and it could not meet the huge domestic and international market demands of red swamp crayfish, boosting the red swamp crayfish aquaculture in China. Right now, China has produced the most amount of crayfish in the world. However, the sustainability of red swamp crayfish culture industry was under threat by many factors, including serious retrogression of red swamp crayfish parent prawn, deterioration of environmental ecosystems and emerging infectious diseases. White spot syndrome virus (WSSV) has been shown to be a pathogen of red swamp crayfish. However, quantitative analysis of WSSV in the tissues of infected red swamp crayfish was not reported. In this report, the epidemic of WSSV in red swamp crayfish from five cities in Hubei province were investigated. More than 80% of red swamp crayfish were WSSV positive when the DNA extracted from gills of red swamp crayfish were subjected to PCR assay. In addition, we also investigated the presence of WSSV antigen in the infected red swamp crayfish by western blot using WSSV-VP28 protein specific serum generated in rabbit. The results showed that a specific band correlated with WSSV-VP28 was present in WSSV positive samples, but not in WSSV negative sample. To better quantify the amount of WSSV in the infected tissues, we established an absolute quantitative real time PCR assay (qRT-PCR) for WSSV using recombinant pGEX-5x-1-VP28. Briefly, plasmid pGEX-5x-1-VP28 containing WSSV-VP28 gene was purified and quantified. Certain amount of pGEX-5x-1-VP28 was diluted serially and used as templates for qRT-PCR using primers specific for VP28 gene amplification. In doing so, a standard curve for the absolute quantification of WSSV was made. The amounts of WSSV genome existed in the tissues were further measured by the qRT-PCR using the same above mentioned primers specific for VP28 gene amplification. The absolute amounts of WSSV genomes in the tissues were obtained by comparison the values of the qRT-PCR with those in the standard curve. Six tissues of red swamp crayfish, including gill, stomach, intestine, hematocyte, hepatopancreas and heart, were sampled and used for WSSV quantification by the absolute qRT-PCR assay. The results showed that the most amounts of WSSV genome (about 108 copies /mg) were detected in the gill, stomach and intestine, followed by hematocyte (about 107 copies /mg), hepatopancreas (about 106 copies /mg). The lowest amount of WSSV was observed in the heart of infected red swamp crayfish, indicating that the replication of WSSV was tissue dependent. The high infection rate of WSSV in the crayfish cultured in the ponds (more than 80% of red swamp crayfish were WSSV positive) indicated that the cultured red swamp crayfish was persistently infected with WSSV. It was reasonable to believe that the disease outbroke when the persistent WSSV was activated under certain conditions, such as poor nutrition or deterioration of environment. Based on the above, we proposed the effective prevention strategies for the WSSV infection in red swamp crayfish culture.