Dataset Information

Integrated qPCR and Staining Methods for Detection and Quantification of Enterocytozoon hepatopenaei in Shrimp Litopenaeus vannamei.

ABSTRACT: Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the optimum annealing temperature was 60 °C for the corresponding relation between the amplification quantitative (Cq) and the logarithmic of the initial template quantity (x), conformed to Cq = -3.2751x + 31.269 with a correlation coefficient R2 = 0.993. The amplification efficiency was 102%. This qPCR method also showed high sensitivity, specificity, and repeatability. Moreover, a microscopy method was developed to observe and count EHP spores in hepatopancreas tissue of EHP-infected shrimp using Fluorescent Brightener 28 staining. By comparing the PTP2-qPCR and microscopy method, the microscopic examination was easier to operate whereas PTP2-qPCR was more sensitive for analysis. And we found that there was a correspondence between the results of these two methods. In summary, the PTP2-qPCR method integrated microscopy could serve for EHP detection during the whole period of shrimp farming and satisfy different requirements for detecting EHP in shrimp farming.

SUBMITTER: Wang L

PROVIDER: S-EPMC7565997 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Integrated qPCR and Staining Methods for Detection and Quantification of Enterocytozoon hepatopenaei in Shrimp Litopenaeus vannamei.

Wang Lijun L Lv Qing Q He Yantong Y Gu Ruocheng R Zhou Bingqian B Chen Jie J Fan Xiaodong X Pan Guoqing G Long Mengxian M Zhou Zeyang Z

Microorganisms 20200907 9

Enterocytozoon hepatopenaei (EHP) is an obligate, intracellular, spore-forming parasite, which mainly infects the gastrointestinal tract of shrimp. It significantly hinders the growth of shrimp, which causes substantial economic losses in farming. In this study, we established and optimized a SYBR Green I fluorescent quantitative PCR (qPCR) assay based on the polar tube protein 2 (PTP2) gene for the quantitative analysis of EHP-infected shrimp. The result showed that the opt ...[more]

PMID: 32906623

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Project description:BACKGROUND: The microsporidian Enterocytozoon hepatopenaei was first described from Thailand in 2009 in farmed, indigenous giant tiger shrimp Penaeus (Penaeus) monodon. The natural reservoir for the parasite is still unknown. More recently, a microsporidian closely resembling it in morphology and tissue preference was found in Thai-farmed, exotic, whiteleg shrimp Penaeus (Litopenaeus) vannamei exhibiting white feces syndrome (WFS). Our objective was to compare the newly found pathogen with E. hepatopenaei and to determine its causal relationship with WFS. RESULTS: Generic primers used to amplify a fragment of the small subunit ribosomal RNA (ssu rRNA) gene for cloning and sequencing revealed that the new parasite from WFS ponds had 99% sequence identity to that of E. hepatopenaei, suggesting it was conspecific. Normal histological analysis using tissue sections stained with hematoxylin and eosin (H&E) revealed that relatively few tubule epithelial cells exhibited spores, suggesting that the infections were light. However, the H&E results were deceptive since nested PCR and in situ hybridization analysis based on the cloned ssu rRNA gene fragment revealed very heavy infections in tubule epithelial cells in the central region of the hepatopancreas in the absence of spores. Despite these results, high prevalence of E. hepatopenaei in shrimp from ponds not exhibiting WFS and a pond that had recovered from WFS indicated no direct causal association between these infections and WFS. This was supported by laboratory oral challenge trials that revealed direct horizontal transmission to uninfected shrimp but no signs of WFS. CONCLUSIONS: The microsporidian newly found in P. vannamei is conspecific with previously described E. hepatopenaei and it is not causally associated with WFS. However, the deceptive severity of infections (much greater than previously reported in P. monodon) would undoubtedly have a negative effect on whiteleg shrimp growth and production efficiency and this could be exacerbated by the possibility of horizontal transmission revealed by laboratory challenge tests. Thus, it is recommended that the PCR and in situ hybridization methods developed herein be used to identify the natural reservoir species so they can be eliminated from the shrimp rearing system.

Project description:It has long been viewed that invertebrates rely exclusively upon a wide variety of innate mechanisms for protection from disease and parasite invasion and lack any specific acquired immune mechanisms comparable to those of vertebrates. Recent findings, however, suggest certain invertebrates may be able to mount some form of specific immunity, termed 'specific immune priming', although the mechanism of this is not fully understood (see Textbox S1). In our initial experiments, either formalin-inactivated Vibrio harveyi or sterile saline were injected into the main body cavity (haemocoel) of juvenile shrimp (Litopenaeus vannamei). Haemocytes (blood cells) from V. harveyi-injected shrimp were collected 7 days later and incubated with a 1:1 mix of V. harveyi and an unrelated gram positive bacterium, Bacillus subtilis. Haemocytes from 'vaccinated' shrimp showed elevated levels of phagocytosis of V. harveyi, but not B. subtilis, compared with those from saline-injected (non-immunised) animals. The increased phagocytic activity was characterised by a significant increase in the percentage of phagocytic cells. When shrimp were injected with B. subtilis rather than vibrio, there was no significant increase in the phagocytic activity of haemocytes from these animals in comparison to the non-immunised (saline injected) controls. Whole haemolymph (blood) from either 'immunised' or non-immunised' shrimp was shown to display innate humoral antibacterial activity against V. harveyi that was absent against B. subtilis. However, there was no difference in the potency of antibacterial activity between V. harveyi-injected shrimp and control (saline injected) animals showing that 'vaccination' has no effect on this component of the shrimp's immune system. These results imply that the cellular immune system of shrimp, particularly phagocytosis, is capable of a degree of specificity and shows the phenomenon of 'immune priming' reported by other workers. However, in agreement with other studies, this phenomenon is not universal to all potential pathogens.

Dataset Information

Integrated qPCR and Staining Methods for Detection and Quantification of Enterocytozoon hepatopenaei in Shrimp Litopenaeus vannamei.

Publications

Integrated qPCR and Staining Methods for Detection and Quantification of <i>Enterocytozoon hepatopenaei</i> in Shrimp <i>Litopenaeus vannamei</i>.

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