槟榔提取物防控凡纳滨对虾虾肝肠胞虫感染效应的研究

Study on the effects of Areca catechu extracts on the prevention and control of enterocytozoon hepatopenaei infection of litopenaeus vannamei

为了评价槟榔提取物对感染虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)的凡纳滨对虾(Litopenaeus vannamei)的总血细胞数、血蓝蛋白浓度和EHP浓度的影响,试验将制备的EHP悬液混入对虾养殖水体中,针对EHP孢壁蛋白基因设计引物,用实时荧光定量PCR检测肝胰腺EHP感染量,当感染量达到约1×10^(3)copies/μg时开始正式试验;将1 g/m L槟榔提取物投入水中,使水体中槟榔提取物浓度保持在1 g/L左右,作为给药组,同时设对照组(不感染不给药)和感染组(感染不给药)。在试验的第1,7,14天分别测定凡纳滨对虾总血细胞数、血蓝蛋白浓度、EHP浓度,并对7,14天的凡纳滨对虾肝胰腺进行Masson染色,观察组织变化。结果表明:在试验第1,7天,感染组血细胞数均显著低于对照组(P<0.05),但与给药组间差异不显著(P>0.05);第14天,感染组血细胞数显著低于对照组和给药组(P<0.05);对照组和给药组间始终差异不显著(P>0.05)。第1天,感染组血蓝蛋白含量显著高于对照组和给药组(P<0.05);第14天,感染组显著高于对照组(P<0.05),与给药组间差异不显著(P>0.05);对照组和给药组间始终差异不显著(P>0.05)。在第7,14天给药组EHP浓度显著低于对照组(P<0.05),感染组EHP浓度分别为给药组的18.00倍和90.52倍;感染组对虾的细胞内寄生了不同生活阶段的孢子虫,细胞出现水肿,第14天在给药组肝胰腺细胞内观察不到孢子。说明槟榔提取物可以作为潜在的治疗肝胰腺微孢子虫病的药物。

In order to evaluate whether Areca catechu extracts could improve shrimp total hemocyte counts,hemocyanin concentration and EHP concentration in Enterocytozoon hepatopenaei(EHP)-infected Litopenaeus vannamei,in the experiment,the prepared EHP suspension was mixed into the shrimp culture water;primers were designed for the EHP wall protein gene,and the amount of EHP infection in the hepatopancreas was detected by quantitative real-time PCR(q PCR).When the infection amount reached about 1×10^(3)copies/μg,the formal trial began;1 g/m L Areca catechu extracts were put into water to keep the concentration of Areca catechu extracts in the water at around 1 g/L.Except the drug group,a control group(no drug and no infection)and an infection group(infection without drug)were set at the same time.On the 1^(st),7^(th),and 14^(th) days of the experiment,the total hemocyte counts,hemocyanin concentration and EHP concentration were measured,and Masson staining was performed on the hepatopancreas of the 7^(th) and 14^(th) day to observe the tissue changes.The results showed that on the first and seventh day of the experiment,the number of blood cells in the infection group was significantly lower than that in the control group(P<0.05).There was no significant difference between the infection group and the drug group(P>0.05).On the 14th day,the number of blood cells in the infected group was significantly lower than that in the control group and the drug group(P<0.05).There was no significant difference between the control group and the drug group(P>0.05).On the first day,the content of hemocyanin in the infection group was significantly higher than that in the control group and the drug group(P<0.05).On the 14th day,the infection group was significantly higher than the control group(P<0.05),and had no significant difference with drug group(P>0.05).There was no significant difference between the control group and the drug group(P>0.05).And the copy number of EHP in the drug group was significantly lower than that in the control group(P<0.05).On the 7^(th) and 14^(th) days,the concentration of EHP in the infection group was 18 times and 90 times that of the drug group,respectively.The sporozoites of different life stages were parasitized in the cells of the prawns in the infection group,and the cells appeared edema.No spores were observed in the hepatopancreatic cells of the drug group on the 14^(th) day.The results suggested that Areca catechu extracts could be used as a potential drug for the treatment of hepatopancreatic microsporidiosis.