2021—2022年虾类偷死野田村病毒(CMNV)流行情况调查

Investigation of the prevalence of covert mortality nodavirus (CMNV) in shrimp from 2021 to 2022

关键词:  对虾病毒性偷死病(VCMD)  偷死野田村病毒(CMNV)  TaqMan探针实时荧光定量  流行病学

Key words:  Viral covert mortality disease (VCMD)  Covert mortality nodavirus (CMNV)  TaqMan probe real-time fluorescence quantitative RT-PCR (TaqMan RT-qPCR)  Epidemiology

摘要:

由偷死野田村病毒(covert mortality nodavirus, CMNV)引起的虾类病毒性偷死病(viral covert mortality disease, VCMD)使对虾养殖产业遭受了严重的损失。为掌握近年CMNV在我国主要养殖虾类中的流行情况，本研究2021—2022年间在全国主要虾类养殖地区开展了CMNV流行病学调查和样品采集工作，利用分子生物学、组织病理学和电镜分析等方法对所采集的样品进行了系统分析。期间，从天津、山东、江苏、海南、湖北及新疆等地共采集1 299份样品，样品种类包括凡纳对虾(Penaeus vannamei)、罗氏沼虾(Macrobrachium rosenbergii)、日本对虾(P. japonicus)、中国对虾(P. chinensis)和克氏原螯虾(Procambarus clarkia)等主要养殖虾类以及养殖池塘中的共生生物。采用TaqMan探针实时荧光定量PCR (TaqMan RT-qPCR)对所采集的样品进行CMNV检测，结果表明，凡纳对虾、罗氏沼虾和日本对虾等主要养殖虾类中均可检测到CMNV阳性，阳性样品采集地包括山东、江苏、海南、新疆、广西和天津等省市；除养殖虾类外，采集的虾类鲜活饵料以及虾类养殖池塘共生生物包括沙蚕(Perinereis aibuhitensis)和卤虫(Artemia sinica)中也可检测到CMNV阳性。2021年和2022年所采集样品中CMNV阳性检出率分别为10.04% (69/687)和11.44% (70/612)。流行病学调查发现，室外池塘养殖对虾中CMNV感染会引起一定程度的累计死亡，室内养殖对虾中CMNV感染主要引起对虾甲壳软化和生长缓慢，通常不会导致其大量死亡。对TaqMan RT-qPCR检测呈阳性的样品进行组织病理和原位杂交分析，患病虾肝胰腺和前中肠盲囊组织中可见嗜酸性病毒包涵体，附肢神经可见空泡化病理损伤，这些发生病理损伤的组织中均有明显CMNV RNA探针紫色杂交信号。本研究结果表明，我国多地养殖虾类以及养殖池塘共生生物中仍存在较高的CMNV阳性检出率，该病毒的流行危害风险仍然较高。建议在甲壳类养殖过程中加强CMNV检测与监测预警，进一步降低其扩散和流行的风险。

Abstract:

Viral covert mortality disease (VCMD) in cultured shrimp is caused by covert mortality nodavirus (CMNV). The infected shrimp mainly die successively in the deep-water areas at the bottom of farming ponds, which is difficult to observe; as such, the term "covert mortality disease" was coined by local farmers. The disease is known as running mortality syndrome by Southeast Asian farmers because a small number of diseased shrimp die daily in the infected ponds, and more continue to die until harvest time. The infected shrimp mostly show symptoms such as carapace softening, hepatopancreas color fading, atrophy, empty stomach, slow growth, etc. In the acute infection stage, the infected shrimp show opaque abdominal muscles. The prevalence of VCMD has caused severe economic losses in the shrimp farming industry over the past decade. CMNV can infect many major cultured shrimp species, including Penaeus vannamei, P. japonicus, P. chinensis, Macrobrachium rosenbergii, and Exopalaemon carinicauda. Moreover, the virus infects the co-inhabiting organisms in shrimp ponds, which makes it difficult to remove from shrimp farming areas due to its wide host range among invertebrates and its transport by many vectors. To elucidate the prevalence of CMNV in the main cultured shrimp species in China in recent years, an epidemiological investigation of CMNV, including clinical sample collection from shrimp and other organisms inhabiting the shrimp ponds, was carried out in the main shrimp culture areas of China from 2021 to 2022, and the collected samples were systematically analyzed using molecular biology, histopathology, and transmission electron microscopy approaches. During this period, 1,299 samples were collected from Tianjin, Shandong, Jiangsu, Hainan, Hubei, and Xinjiang and included P. vannamei, M. rosenbergii, P. japonicus, P. chinensis, Procambarus clarkia, and other organisms living in shrimp culture ponds. The collected samples were analyzed using CMNV TaqMan probe real-time quantitative reverse transcription PCR (TaqMan RT-qPCR). The results showed that CMNV could be detected in the main cultured shrimp species, including P. vannamei, M. rosenbergii, and P. japonicus. A percentage of the samples collected from Shandong, Jiangsu, Hainan, Xinjiang, Guangxi, and Tianjin were observed to be CMNV-positive in the TaqMan RT-qPCR assays. In addition to cultured shrimp, CMNV was detected in organisms living in the shrimp ponds, such as Artemia collected in 2021 and Perinereis aibuhitensis collected in 2022. The CMNV-positive rate of samples collected in 2021 was 10.04% (69/687), and that of samples collected in 2022 was 11.44% (70/612). Epidemiological investigation showed that CMNV infection in outdoor pond culture usually caused a certain degree of cumulative death, whereas CMNV infection in indoor culture mainly caused the softening and reduced growth of infected shrimp and did not cause a large number of infected shrimp to die. High CMNV loads were detected in samples of Artemia used as fresh bait for shrimp and in samples of P. aibuhitensis living in shrimp pond soils. Accordingly, a high CMNV infection rate was observed in cultured P. vannamei feeding on CMNV-positive Artemia and in that farmed in ponds containing CMNV-positive P. aibuhitensis. The results indicated that CMNV vectors such as Artemia and P. aibuhitensis may pose a high risk of CMNV transition, threatening the health of the shrimp in the culture system. Recent studies on the pathogenesis of CMNV infection indicated that the virus mainly infected epithelial tissues, nerve tissues, and connective tissues in the hepatopancreas, appendages, mid-gut, gills, eye stalks, and nerves of shrimp. Therefore, histopathological and in situ hybridization analyses were performed on samples identified as CMNV-positive using TaqMan RT-qPCR in this study. Eosinophilic virus inclusion bodies were observed in the hepatopancreas and foremidgut tissues, and pathological lesions were observed in the appendicular nerves of diseased Penaeus shrimp. Moreover, obvious purple hybridization signals from the CMNV RNA probe were seen in tissues with lesions, which further confirmed the positive detection results for CMNV in shrimp samples obtained using TaqMan RT-qPCR in this study. The results of this study showed that, compared with previous years, there was still a high CMNV-positive detection rate in many areas of crustacean culture in 2022, and the epidemic risk of this virus was still high. Multiple CMNV-positive results were obtained in the co-inhabiting and feed organisms in crustacean farming ponds. These results remind fishery authorities and stakeholders in the prawn breeding industry to further strengthen the monitoring and early detection of CMNV to prevent and control the spread of the virus in seedlings, reduce the risk of epidemic hazards, and promote the sustainable and healthy development of the crustacean aquaculture industry.