凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(EHP)的荧光定量PCR检测

Detection and Quantification of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and Enterocytozoon hepatopenaei (EHP) of Litopenaeus vannamei by Real-Time PCR

2013年,河北、天津等地区养殖的凡纳滨对虾(Litopenaeus vannamei)育苗期出现死苗、出苗率低的情况,生产上,仔虾个体大小差异较大,造成了严重损失。本研究采用荧光定量PCR方法(Real-time PCR)对天津大港地区采集的108尾凡纳滨对虾仔虾样品进行单尾病原检测。结果显示,传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus,IHHNV)和虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)均有检出。IHHNV阳性检出率100%,每微克对虾组织DNA的病毒拷贝数为10~3–10~7,且个体较大的样品(1.2–2.0 cm)携带病毒拷贝数偏高;EHP阳性检出率为49.1%,每微克对虾组织DNA的拷贝数为10~3–10~5,且集中于个体较小样品(0.7–1.1 cm)。对IHHNV和EHP阳性凡纳滨对虾样品进行生物学体长与病毒载量指数相关性分析,显示IHHNV载量指数与对虾生长速率呈正相关,虾组织IHHNV平均载量达8.51×10~4 copies/μg DNA,为较高的感染水平;EHP的载量与对虾生长速率呈负相关关系,与较大个体阳性检出率较低相对应,虾组织EHP平均载量达到2.19×10~4 copies/μg DNA,为较高的感染水平。由此,该批凡纳滨对虾仔虾患病为IHHNV和EHP的混合感染所致,本研究数据为IHHNV和EHP病原混合感染流行情况及其对养殖育苗期仔虾生长的影响提供科学依据。

Infectious hypodermal and hematopoietic necrosis virus （IHHNV） is one of the pathogens causing the significant diseases of crustaceans listed by the OIE （Office internaptional despizooties） Manual of Diagnostic Tests for Aquatic Animals. Infection with IHHNV does not produce any pathognomonic gross clinical signs, although runt-deformity syndrome （RDS） has been noted in infected juvenile Litopenaeus vannamei and Penaeus monodon. Enterocytozoon hepatopenaei （EHP） was first found and isolated in Thailand in 2009, which caused slow growth of P. monodon. IHHNV and EHP were found in the samples of L. vannamei postlarvae which were collected from hatchery farms in Tianjin and Hebei Province in 2013. The diseased shrimps showed some clinical signs with high mortality, slow growth and difference of individual growth rate. The IHHNV and EHP individual detection was conducted on these samples using the real-time PCR technique. In total, 108 out of 108 （100%） L. vannamei postlarvae samples were IHHNV positive, and 53 out of 108 （49.1%） L. vannamei postlarvae samples were EHP positive. Positive samples contained approximately 10^3–10^7 copies/（μg DNA） of IHHNV and big size of individual shrimp carried higher number of virus copies. Positive samples contained approximately 10^3–10^5 copies/（μg DNA） of EHP and mainly the small sized individual shrimp. The analysis showed that the IHHNV loads were positively correlated with the shrimp growth rate. IHHNV loads above 8.51×10^4 copies/μg DNA represented a high risk level. EHP loads were negatively correlated with the shrimp growth rate. EHP loads above 2.19×10^4 copies/μg DNA represented a high risk level. Our study showed that the diseased L. vannamei postlarvae samples occurred in the hatchery farms of Tianjin and Hebai Province infected with shrimp pathogens of IHHNV and EHP. Data provides scientific basis on the effects of pathogens infection on the growth of cultured penaeid shrimp.