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The Causes and Prevention of EHP

A Brief introduction of EHP

 

Enterocytozoon hepatopenaei

According to FAO statistics, the scale of global shrimp farming has reached 4.2 million metric tons in 2011. However, after the outbreak of EMS in China in 2009, production fell by 6% between 2011 and 2013. As of 2016, although the key shrimp farming countries have gradually recovered from the impact of EMS, and global production may grow at a rate of 4.2% (GOAL, 2016 forecast), shrimp diseases are still the gravest issue and challenge in the world. In addition to various bacterial and viral diseases that have prevailed in the past, we are also seeing several new diseases in recent years, among which the Enterocytozoon hepatopenaei (EHP) infection of marine shrimp has become the biggest concern. (Yang Minghua, Zheng Jinhua, Chen Zizhen, 2016.)

Classified very close to fungi, EHP is an obligate intracellular parasite. In 2004, it was first reported that in Thailand, an unknown microsporidian inhibited the growth of Penaeus monodon; Tourtip et al. then isolated the pathogen from the slow-growing monodon for further description and naming. More EHP infections were later found among cultured Litopenaeus vannamei and Penaeus stylirostris (Tang et al., 2015). The difference between EHP and other microsporidians is that it hurts the tubule epithelial cells of the hepatopancreatic tissues of a shrimp, thereby interfering with its normal nutrient absorption. When the infected hepatopancreatic tissue is sectioned and colored with Hematoxylin and Eosin (H&E) staining, inclusion bodies in the cytoplasm can be identified, while oval or elliptical spores of 1.1±0.2~0.6-0.7±0.1 μm may cluster. Sometimes, we can see spores moving from lysed cells into the lumen. Thanks to a penetrating electric display, a single nucleus can be observed in the spore. Under the TEM, it can be observed that there is a single nucleus in the spore, a posterior vacuole, and anchoring disk attached to the polar filament. Five to six polar filaments are seen wrapping around the thick electron-dense wall. (Tourtip et al., 2009).

 

EHP Stain Examination

Photomicrographs of Enterocytozoon hepatopenaei tubule epithelial cells of the hepatopancreas of Penaeus monodon.

 
 

(A) H&E-stained smear of hepatopancreatic tissue showing numerous microsporidian spores. (B) Fresh preparation of microsporidian spores from a Percoll gradient.

 
 

(C and D) Hepatopancreatic tissue sections showing acidophilic, granular inclusions in the cytoplasm of tubule epithelial cells.

 

 

(E) Semi-thin section of hepatopancreatic tissue showing early and late plasmodia (inset a) and mature spores (inset b) in the cytoplasm of tubule epithelial cells. Some spores show unstained spots that represent their concave surfaces at one end (inset b).

  • (A) Hematoxylin stain;
  • (C) Trichrome stain;
  • (D) H&E stain;
  • (E) Toluidine blue stain;
  • (ePm) early plasmodium;
  • (lPm) late plasmodium. (Tourtip et al., 2009)

 

Microsporidia infection-host and mode of transmission

Currently, EHP infections are seen in China, Vietnam, Thailand, Malaysia, Indonesia, India, Brunei, and possibly also in the Philippines and Mexico. The prevalence rate in Thailand is 49% (Flegel et al., 2014). In general, most of the epidemic areas are in Asia, which may be due to the nature of the disease or the stricter biosecurity and epidemic prevention measures in other regions. In most cases, EHP mainly spreads via imported live baits (such as Nereis Spp.) and live shrimp from pandemic regions. Since Nereis Spp. production areas often overlap with those of shrimp farming, Nereis Spp. circulation may easily trigger cross-infections of shrimp diseases. In Thailand, whether it was imported SPF vannamei PL or locally bred PL from imported SPF vannamei brood stock that was used, EHP was not detected before stocking. However, a while later, infections were confirmed in the ponds, which means the pathogen may have pre-existed locally and thus easily leading to horizontal infections. Scholars also suspect that EHP may be cultivated locally and has been associated with local monodon farming for several years (Rajendran et al., 2016; Tangprasittipap et al., 2013). It is proven that horizontal infection of EHP may be due to ingestion of sick shrimp or symbiosis (Han, 2016; Tang et al., 2016; Tangprasittipap et al., 2013).

Infection Methods

Horizontal

Live bait, digestion of sick shrimp

Vertical

Not verified yet

 

EHP and Slow Growth

Since EHP is parasitic in the lumen of the hepatopancreatic epithelial cells of the shrimp, it affects the normal absorption and storage of nutrients by the liver and pancreas. Although it does not necessarily lead to death of the shrimp, it is generally recognized as the main cause of slow growth. Scholars believe that EHP has been common in Asia for many years and has been neglected because EMS, which has higher mortality and is more prevalent, was more in the focus.

 

While the strategy to deal with early death has been effective in recent years, the surviving shrimp’s growth was very limited and uneven. Not knowing the cause of the disease would bring about a dilemma of whether to harvest or not and would eventually result in serious losses and indirectly lead to lower vannamei exports from Asia during those years. Liu et al. (2015) conducted real-time quantitative PCRs to verify the EHP small subunit ribosomal DNA (SSU rDNA) in the DNA of hepatopancreatic tissues from three batches of samples collected from vannamei shrimp in Jiangsu, Hainan and Shandong, China. The results showed that the concentration of EHP (copies/ng HP DNA) is negatively correlated with the growth rate of vannamei. (Yang Minghua, Zheng Jinhua, Chen Zizhen, 2016.)

 

EHP Preventive Measures

Shrimp infected with EHP do not show visible symptoms, which may delay treatment and spread the epidemic. Currently there is no effective method to treat EHP. Once infection is confirmed, very often it will stay, and the only way to deal with it is epidemic control and the implementation of biological preventive measures from breeding to farming.

 

  • PL management
    • Confirm with PCR tests that the PL is not infected.
    • Ensure no EHP contamination of live bait during selection stage.
  • Instrument disinfection
    • The equipment in the field and the nursery must be disinfected before use.
  • Environment and water quality management
    • Increase sewage discharge capacity to avoid deterioration of the water quality in the pond.
    • Add probiotics to the water to avoid proliferation of pathogens or bacteria.
    • Avoid inappropriate use of medicine, lest it cause hepatopancreatic damage.
  • Disease management
    • Use Grobest functional feed at the early stage of stocking to enhance immunity.
    • Dead shrimp should be removed as possible to avoid cross-infection.
    • Use high-quality SPF shrimp PL (base on PCR testing protocols).
  • Grobest functional feed:
    • enhances intestinal immunity, protects intestinal tract, increases palatability and resistance to diseases;
    • stimulates the shrimp’s non-specific immune function, enhances non-specific immunity, reduces the chance of viral disease infection, and has apparent effects on the prevention of shrimp diseases;
    • contains sufficient vitamins and amino acids which can supplement nutrients physiologically required for the shrimp, satisfying their demand for enzymes and trace elements, promote metabolism, and help maintain the healthy growth and development of shrimp;
    • contains selected glucans to improve the structure of intestinal flora, promote metabolism, protect cells from free radicals, maintain normal cell metabolism, and enhance resistance;
    • promotes the production of digestive enzymes, can enhance digestion, and has anti-oxidant function to increase growth efficiency;
    • increases the resistance of animals to diseases and the ability of cell recovery which can also prevent diseases caused by stress.

 

Application methods

  • Water quality indexes are ammonia < 0.3 ppm, nitrite < 1 ppm, DO ≥ 4 ppm, pH 7.5-8.5. When the water quality is abnormal, use Grobest's water quality treatment agent, such as PSB and EM bacteria to adjust the water quality; apply it with functional feed.

  • Usage instructions
    • During nursery period (< DOC 30): 15 consecutive days
    • Prevention: 5 to 7 days of consecutive use every two weeks
    • Before high-risk season: 15 days of continuous use before outbreak season or environmental changes