Microsporidian in Shrimp – Enterocytozoon hepatopenaei (EHP)
The causative agent of EHP in shrimp is Enterocytozoon hepatopenaei. It belongs to the family Enterocytozoonidae and phylum Microsporidia. It is closely related to fungi. Some of the similar characteristics with fungi are presence of chitin in cell wall, presence of intranuclear mitosis (Dunn and Smith, 2001) and lack of flagella. It is the smallest and simplest eukaryote. The size of the spore is about 1.1 ± 0.2 by 0.6-0.7 ± 0.1um (Tourtip et al., 2009).
Figure 1: Electron micrograph of spores of EHP (Source: Chaijarasphong et al., 2020)
It is surrounded by a double-layered spore wall which protects it from the harsh environment. The spore wall protein is responsible for binding with the surface of the host cell. The polar tube act as a transport tube that deliver the spore content known as sporoplasm into the cytoplasm of host cell. The extrusion of polar tube is suggested to be triggered by certain environmental condition for example changes in pH or ion concentrations (Williams, 2009).
Shrimp infected with EHP does not show any distinctive clinical sign. It can be suspected by retarded growth and increase in size variation (Figure 2). EHP affect the hepatopancreas (HP) of the shrimp making it more susceptible to white feces syndrome (WFS) and acute hepatopancreatic necrosis disease (AHPND). There are a few reports found EHP associated with WFS (Ha et al., 2010) or AHPND (Sanguanrut et al., 2018). Hepatopancreas is an important organ in shrimp which involve in digestion, absorption and producing immunity.
Figure 2: Retarded growth of Penaeus vannamei observed during ~90 days of culture (Source: Rajendran et al., 2016)
EHP belongs to the Enterocytozoon-group Microsporidia (EGM) (Figure 3). This group comprise of several other genera including Enterospora, Desmozoon, Obruspora, Nucleospora and Enterocytospora. They infect a wide range of hosts including human, mammals and aquatic animals. Enterospora is reported in gilt-head bream, brown crab, hermit crab and artemia.
Figure 3: Phylogeny and host range of known members of the EGM. Except for E. bieneusi infecting humans, terrestrial mammals, and birds, all other members of the EGM infect aquatic hosts. (Source: Stentiford et al., 2019)
Life cycle of microsporidia can be divided into three phases: the infective, the proliferative and the spore-forming phase. The infective phase is the only phase that can survive without the host (Keeling, 2009). It can be viable for up to 6 months in fecal pellet and dried carcasses as well as 1 year in cold aqueous condition (Kramer, 1970). Microsporidia depend fully on the host cell nutrient-rich environment to proliferate (Williams, 2009). Some species are reported to takes 3-4 days for maturation of spore and completion of full life cycle (Leitch and Ceballos, 2008; Wasson and Barry, 2003).
The infective spore punctures the plasma membrane of the host cell with its polar tube releasing sporoplasm inside the cytoplasm of the cell. Sporoplasm proliferate inside the cells forming branched plasmodia. Spore extrusion precursor forms prior to break down of the plasmodium cytoplasm into sporoblasts. The infected HP epithelial cell swells and rupture releasing the mature spore in the hepatopancreas or discharge to the environment via feces. Healthy shrimp get infected by accidental ingestion of spore from feces or cannibalism.
Figure 4: Schematics illustrating the life cycle and transmission routes of EHP. (Source: Chaijarasphong et al., 2020)
The route of transmission to the epithelial hepatopancreatic cell was suggested through the posterior stomach chambers (PSC). PSC of penaeid shrimp contain a filtration structure called gastric sieve (GS). The GS serves as a selective filter that excludes large partially digested food particles but allows smaller particles and soluble materials to enter hepatopancreatic ducts that conduct them into the shrimp hepatopancreas (HP), where further digestion and absorption of nutrients takes place. The GS sieve pore diameter is approximately 0.2–0.7 µm in size, indicating a size exclusion limit of substantially less than 1 µm (Pattarayingsakul et al., 2019). The size of the infective spore is small enough to pass through the sieve and travel to the HP.
The spore wall protein might be involved in host cell recognition. It was demonstrated that the spore wall protein could bind with heparin (Jaroenlak et al., 2018). The main role of heparin is act as a blood anticoagulant (Linhardt, 2003). Heparin is extensively studied in vertebrate. The distribution of heparin in shrimp was not known but heparin is successfully extracted from the cephalothorax in the red-spotted shrimp P. brasilliensis and Pacific white shrimp P. vannamei (Brito et al., 2014; Dietrich et al., 1999). Stomach, hepatopancreas, heart, gut and gills are located at the cephalothorax.
Some of the possible carrier of EHP is reported to be PCR positive such as polychaetes, artemia, mollusk and squid.
Microscopic examination of EHP require oil immersion as the spore is very small size. Some of the drawback of microscopic examination is it might be hard to identify without specific staining method when the level of infection is low and it might go undetected due to uneven distribution of EHP in HP. Squash mount of HP without staining can be observed under phase contrast microscope. Tissue smears can be prepared by 3 different methods. First, it can be fixed with absolute methanol for 15 minutes and stained with Giemsa. Second, it can be fixed with 5% neutral buffered formalin for 15 minutes and stained with haematoxylin and eosin (H&E). Last, there is a specific stain used for microsporidia, the tissue smear fixed with 100% ethanol for 10 minutes, stained with Trichome blue for 90 minutes, dipped in acid ethanol 90% for 1 to 3 seconds, rinse with 95% ethanol, place in two additional changes of 95% ethanol for 5 minutes each, place in 100% ethanol for 10 minutes, place in two changes of xylene-s for 10 minutes each.
Figure 5: Light microscopic observations on the spores of EHP recovered from L . vannamei. A. Smear preparation of infected hepatopancreas stained with Giemsa showing EHP spores. B. Smear preparation stained with H&E (arrows indicate spores) (Source: Rajendran et al., 2016)
Figure 6: Two photomicrographs of HP smears stained with H&E and showing EHP plasmodia and spores. (Source: Chaijarasphong et al., 2020)
Figure 7: EHP spores in the white faecal samples on modified trichome staining. (Source: Praveena, 2018)
There is 2 methods reported for molecular examination which SSU-based PCR (Tangprasittipap et al., 2013) and SWP-based PCR (Jaroenlak et al., 2016). SSU-based PCR is the most extensively used method. This method has limited resolution for species definition. False positive test results of EHP are reported due to the primer has high sequence identity with other aquatic microsporidians. This method is not suitable for screening shrimp’s feces, feeds and pond water as it might be contaminated by closely-related EGM. SWP-based PCR is designed to detect the spore wall protein (EhSWP1) gene. It helps to distinguish EHP from other closely-related EGM. It did not cross react with DNA from closely-related EGM. Sample preparation for molecular examination of EHP have to be done correctly to avoid false negative. EHP distribute unevenly in the HP. Whole HP should be homogenize and follow by the removal of sub sample for DNA extraction. Combination of histology and molecular technique are required for confirmation of EHP.
EHP can be controlled by manipulating the pH. Study was found high pH stimulate spore extrusion. Purified spore was incubated in pH 4, 7 and 9 buffer which promote 5, 10 and 90% of spore extrusion, respectively (Chaijarasphong et al., 2020). Commercial lime is recommended for pond preparation to stimulate spore extrusion before stocking of animal. EHP has been found to be killed by high temperature (Aldama-Cano et al., 2018). Live or fresh shrimp feeds should be frozen at -20°C for at least 48 hours. Better treatment by pasteurized at 70°C for 15 minutes.
There is limited research done for Enterocytozoon hepatopenaei. Some question to be answered:
- Is SWP-PCR based method a reliable method for detection of EHP? Any false positive from other closely related microsporidia?
- What is the best practical way of diagnosing EHP?
- What is the environmental condition that trigger spore extrusion?
- How to control EHP?
Chaijarasphong T., Munkongwongsiri N., Stentiford G. D., Aldama-Cano D. J., Thansa K., Flegel T. W., et al. 2020. The shrimp microsporidian Enterocytozoon hepatopenaei (EHP): Biology, pathology, diagnostics and control. Journal of Invertebrate Pathology, 107458.
Williams B. A. 2009. Unique physiology of host-parasite interactions in microsporidia infections. Cell Microbiology. 11: 1551-1560.
Rajendran K.V., Shivam S., Praveena P.E., Sahayakajan J.J., Kumar T.S., Avunje S., et al. 2016. Emergence of Enterocytozoon hepatopenaei (EHP) in farmed Penaeus (Litopenaeus) vannamei in India. Aquaculture. 454: 272-280.
Stentiford G. D., Bass, D. & Williams B. A. P. 2019. Ultimate opportunists—the emergent Enterocytozoon group microsporidia. PLoS Pathogens. 15(5): e1007668.
Keeling P. J. 2009. Five questions about Microsporidia. PLoS Pathogens. 5: e1000489.
Kramer J. P. 1970. Longevity of microsporidian spores with special reference to Octosporea muscaedomesticae Flu. Acta Protozoologica. 8: 217-224.
Leitch G. J., Ceballos C., 2008. Effects of host temperature and gastric and duodenal environments on microsporidia spore germination and infectivity of intestinal epithelial cells. Parasitology Research 104: 35–42.
Wasson K., Barry P. A., 2003. Molecular characterization of Encephalitozoon intestinalis (Microspora) replication kinetics in a murine intestinal cell line. Journal of Eukaryotic Microbiology. 50: 169–174.
Pattarayingsakul W., Pudgerd A., Munkongwongsiri N., Vanichviriyakit R., Chaijarasphong T., Thitamadee S. et al. 2019. The gastric sieve of penaeid shrimp species is a sub-micrometer nutrient filter. Journal of Experimental Biology. 222: jeb199638.
Praveena P.E., Bhuvaneswari T., Krishnan A.N., Jagadeesan V., Rajan J.J.S., Jithendran K.P. 2018. An improved microscopic method for the rapid diagnosis of emerging microsporidian parasite, Enterocytozoon hepatopenaei in shrimp farms. Current Science. 115: 758-762.
Jaroenlak R., Sanguanrut P., Williams B.A.P., Stentiford G.D., Flegel T.W., Sritunyalucksana K. et al. 2016. A Nested PCR Assay to Avoid False Positive Detection of the Microsporidian Enterocytozoon hepatopenaei (EHP) in Environmental Samples in Shrimp Farms. PLOS one. 11: e0166320.
Jaroenlak R., Boakye D. W., Vanichviriyakit R., Williams B. A. P., Sritunyalucksana K., Itsathitphaisarn O. 2018. Identification, characterization and heparin binding capacity of a spore-wall, virulence protein from the shrimp microsporidian, Enterocytozoon hepatopenaei (EHP). Parasites & Vectors. 11:177.
Tangprasittipap A., Srisala J., Chouwdee S., Somboon M., Chuchird N., Limsuwan C., et al. 2013. The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei. BMC Veterinary Research. 9: 139.
Aldama-Cano D. J., Sanguanrut P., Munkongwongsiri N., Ibarra-Gámez J. C., Itsathitphaisarn O., Vanichviriyakit R., et al. 2018. Bioassay for spore polar tube extrusion of shrimp Enterocytozoon hepatopenaei (EHP). Aquaculture. 490: 156-161.
Linhardt R. J. 2003. 2003 Claude S. Hudson Award Address in Carbohydrate Chemistry. Heparin: structure and activity. Journal of Medicinal Chemistry. 46: 2551–2564.
Brito A. S., Cavalcante R. S., Palhares L. C., Hughes A. J., Andrade G. P., Yates E. A., et al. 2014. A non-hemorrhagic hybrid heparin/heparan sulfate with anticoagulant potential. Carbohydrate Polymers. 99: 372–378.
Dietrich C. P., Paiva J. F., Castro R. A. B., Chavante S. F., Jeske W., Fareed J., et al. 1999. Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis. Biochimica et Biophysica Acta – General Subjects. 1428: 273–283.
Dunn A. M., Smith J. E. 2001. Microsporidian life cycles and diversity: the relationship between virulence and transmission. Microbes and Infection. 3: 381-388.
Tourtip S., Wongtripop S., Stentiford G. D., Bateman K. S., Sriurairatana S., Chavadej J. et al. 2009. Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships. Journal of Invertebrate Pathology. 102: 21-29.
Ha N. T., Ha D. T., Thuy N.T., Lien V. T. K. 2010. Occurrence of microsporidian Enterocytozoon hepatopenaei in white feces disease (WFS) of cultured black tiger shrimp (Penaeus mondon) in Vietnam. Aquatic Animal Health. https://aquahealth.wordpress.com.