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. 2022 Sep 23:12:1013016.
doi: 10.3389/fcimb.2022.1013016. eCollection 2022.

A modification of nested PCR method for detection of Enterocytozoon hepatopenaei (EHP) in giant freshwater prawn Macrobrachium rosenbergii

Yuan Wang  1   2   3 , Jinyang Zhou  1   2   3 , Menghe Yin  1 , Na Ying  1 , Yang Xiang  1 , Wenchang Liu  1 , Junqiang Ye  4 , Xincang Li  1 , Wenhong Fang  1 , Hongxin Tan  2   3
Affiliations

A modification of nested PCR method for detection of Enterocytozoon hepatopenaei (EHP) in giant freshwater prawn Macrobrachium rosenbergii

Yuan Wang et al. Front Cell Infect Microbiol. .

Abstract

The microsporidian Enterocytozoon hepatopenaei (EHP) has become a critical threat to the global shrimp aquaculture industry, thus necessitating early detection by screening. Development of a rapid and accurate assay is crucial both for the active surveillance and for the assessment of shrimp with EHP infection. In the present study, a distinct strain of E. hepatopenaei (EHP Mr ) was found in Macrobrachium rosenbergii. The SWP1 gene analysis revealed it was a new genotype that differed with the common strain isolated from the Litopenaeus vannamei (EHP Lv ). A nested SWP-PCR method was modified to fix the bug that the original inner primers could not recognize the EHP Mr strain. The redesigned inner primers successfully amplified a product of 182 bp for both the EHP Mr strain and the EHP Lv strain. The new primers also had good specificity and high sensitivity, which may serve as an alternative for EHP genotyping. This study provided a method for detection of EHP in the biosecurity of Macrobrachium rosenbergii farming, and the developed protocol was proposed for the routine investigation and potential carrier screening, especially for molecular epidemiology.

Keywords: Enterocytozoon hepatopenaei; Macrobrachium rosenbergii; microsporidia; nested PCR; spore wall protein.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Alignment of partial nucleotide sequences of the SWP1 gene of EHP Lv , EHP Mr , and reference EHP (MG015710). The nucleobases with white differ from the consensus. The numbers on the right indicate the nucleotide position.
Figure 2
Figure 2
Alignment of partial protein sequences of the SWP1 from EHP Lv , EHP Mr , and reference EHP (GenPept accession nos. AVQ09707). The amino acids with white differ from the consensus. The numbers on the right indicate the amino acid position in the published sequence.
Figure 3
Figure 3
Phylogenetic tree of SWP1 gene of EHP isolates with other microsporidia. Nosema pernyi is used as the outgroup. Bootstrap values are indicated on the branches.
Figure 4
Figure 4
Nested PCR for the detection of the two EHP stains in Litopenaeus vannamei and Macrobrachium rosenbergii by using the SWP2F/2R primers and SWP2F′/2R′ primers, respectively. Lanes V1~V3: the hepatopancreatic DNA of EHP Lv -infected Litopenaeus vannamei; Lanes R1~R3: the hepatopancreatic DNA of EHP Mr -infected Macrobrachium rosenbergii; M: molecular weight marker; +: positive control, SWP1 gene plasmid DNA; v-: negative control, the hepatopancreatic DNA of healthy Litopenaeus vannamei, R-: negative control, the hepatopancreatic DNA of healthy Macrobrachium rosenbergii.
Figure 5
Figure 5
Comparison of sensitivity of the inner primers (SWP2F/2R) and new inner primers (SWP2F′/2R′) to amplify the SWP1 gene. M: molecular weight marker; 1-9:1×108 -1×100 copies of 10-fold dilutions of pGEM-SWP1; N: negative control, DNA samples of healthy P. vannamei.
Figure 6
Figure 6
Validation of the specificity of improved nested PCR detection. M: molecular weight marker; lane 1, Enterospora epinepheli; lane 2, Nucleaspora hippocampi; lane 3, Enterocytospora artemiae; lane 4, Ameson portunus; lane 5, Potaspora sp.; +: positive control.
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References

    1. Azad M. A. K., Islam S. S., Amin M. N., Ghosh A. K., Hasan K. R., Bir J., et al. . (2021). Production and economics of probiotics treated Macrobrachium rosenbergii at different stocking densities. Anim. Feed Sci. Technol. 282, 115125. doi: 10.1016/j.anifeedsci.2021.115125 - DOI
    1. Behera B. K., Das A., Paria P., Sahoo A. K., Parida P. K., Abdulla T., et al. . (2019). Prevalence of microsporidian parasite, Enterocytozoon hepatopenaei in cultured pacific white shrimp, Litopenaeus vannamei (Boone 1931) in West Bengal, East coast of India. Aquacult. Int. 27 (2), 609–620. doi: 10.1007/s10499-019-00350-0 - DOI
    1. Biju N., Sathiyaraj G., Raj M., Shanmugam V., Baskaran B., Govindan U., et al. . (2016). High prevalence of Enterocytozoon hepatopenaei in shrimps Penaeus monodon and Litopenaeus vannamei sampled from slow growth ponds in India. Dis. Aquat. Org. 120 (3), 225–230. doi: 10.3354/dao03036 - DOI - PubMed
    1. Bonneaud C., Longdon B. (2020). Emerging pathogen evolution using evolutionary theory to understand the fate of novel infectious pathogens. EMBO Rep. 21 (9), e51374. doi: 10.15252/embr.202051374 - DOI - PMC - PubMed
    1. Chaijarasphong T., Munkongwongsiri N., Stentiford G. D., Aldama-Cano D. J., Thansa K., Flegel T. W., et al. . (2021). The shrimp microsporidian Enterocytozoon hepatopenaei (EHP): Biology, pathology, diagnostics and control. J. Invertebr. Pathol. 186, 107458. doi: 10.1016/j.jip.2020.107458 - DOI - PubMed

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