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

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 (EHPMr) 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 (EHPLv). A nested SWP-PCR method was modified to fix the bug that the original inner primers could not recognize the EHPMr strain. The redesigned inner primers successfully amplified a product of 182 bp for both the EHPMr strain and the EHPLv 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.

Bacteriophage and non-pathogenic Vibrio to control diseases in shrimp aquaculture

“…The DNA of shrimp infected with WSSV was used as the template and the target gene VP28 was amplified by PCR to obtain the target fragment of 140 bp (F-AGGTGTGGAACAACACATCAAG, R-TGCCAACTTCATCCTCATCA) [ 66 ]. The DNA of shrimp infected with EHP was used as the template and the target gene SWP1 was amplified by PCR, the primer (F- 5’-GCAGAGTGTTGTTAAGGGTTTAAG-3’, R- 5’-GCTGTTTGTCWCCAACTGTATT-3’) were designed to target 182 bp [ 67 ]. For this, we extracted DNA from live and moribund shrimp, conducted PCR, gel electrophoresis, and then gel documentation to observe the band's presence to confirm shrimp infection in the collected samples.…”

Enterocytozoon hepatopenaei Infection in Shrimp: Diagnosis, Interventions, and Food Safety Guidelines

“…Moreover, a modified method of nested PCR to detect EHP in Macrobrachium rosenbergi , giant freshwater prawn was carried out in which the primers were redesigned to detect the distinct strain of EHP. This is because the former primers could not amplify the EHP SWP1 gene; thus, the nested SWP-PCR method was altered, and the new primers showed high specificity and sensitivity [ 16 ]. Overall, this study’s method was proposed to be beneficial for investigating EHP mutants in epidemiological studies, and SWP1 gene mutation allows for a better understanding of the molecular mechanisms by which EHP adapts to diverse hosts.…”

Enterocytozoon hepatopenaeiProliferate inProcambarus clarkii: A Warning for Crayfish and Shrimp Aquaculture

“…EHP can be transmitted horizontally though water (Salachan et al, 2017), feces (Singh and Singh, 2018) and carcasses (Tangprasittipap et al, 2013; Jang et al, 2022). Besides L. vannamei and P. monodon , EHP was found proliferate in blue shrimp ( Penaeus stylirostris ) (Tang et al, 2015), giant river prawn ( Macrobrachium rosenbergi ) (Wang et al, 2022) and dragonfly ( Anax parthenope , Pantala flavescens , and Ischnura senegalensis ) (Kumar Dewangan et al, 2023). EHP can also be transmitted between live foods and other animals living in the same waters, such as polychaetes (Desrina et al, 2020), brine shrimp ( Artemia salina ) (Karthikeyan and Sudhakaran, 2020), false mussels ( Mytilopsis leucophaeata ) (Munkongwongsiri et al, 2022), marine crabs (Mani et al, 2022) and some macrofauna (Wan Sajiri et al, 2023).…”