The results suggest that shrimp feces may be a route of infection.
This study evaluated the effects of different salinities on infection by the intracellular microsporidian Enterocytozoon hepatopenaei (EHP) in whiteleg shrimp under experimental conditions. The results showed that shrimp feces could be a route of EHP infection occurring at salinities as low as 2 ppt but the incidence and severity of EHP infection were higher at 30 ppt.
Hepatopancreatic Microsporidiosis (HPM), caused by the intracellular microsporidia Enterocytozoon hepatopenaei (EHP), has been reported in several species of penaeid shrimp, including black tiger shrimp and whiteleg shrimp. EHP has been reported in various regions, including Asian countries such as China, Indonesia, Malaysia, Vietnam, Thailand, and India. Recently, EHP has also been reported in Venezuela, located in the western hemisphere.
EHP causes lesions in the hepatopancreatic (HP) tubule epithelial cells, and its main clinical signs are growth retardation leading to increased size variation. White fecal filaments floating on the surface of pond water and the presence of white shrimp in the gastrointestinal (GI) tract in these ponds have also been associated with EHP. In advanced stages of the disease, EHP-infected shrimp often have soft shells, lethargy, reduced feed intake, empty midguts, and chronic mortality.
In countries where EHP has been reported, such as India, China, Vietnam, and Venezuela, shrimp farming is carried out in a wide range of environmental conditions, including coastal waters, estuarine waters, and inland waters. For example, in some states in the eastern part of India, such as Andhra Pradesh, ponds are filled with borehole water mixed with estuarine water, resulting in salinities in the grow-out ponds ranging from 0 to 30 ppt with an average salinity of around 10 ppt. In contrast , in states located in the western part of the country, such as Gujarat, salinities in pond water can range from 30 to 44 ppt. In northwestern Venezuela, some shrimp farms are located around Lake Maracaibo, where salinities range from 2 to 5 ppt, while in northeastern Venezuela, shrimp farms are located in marine environments where salinities range from 20 to 40 ppt, and EHP has been reported in both high and low salinity environments. In both countries, the prevalence of EHP appears to be higher in high salinity environments, but to date no studies have evaluated the possible relationship between salinity and EHP presence.
Research setup
Bioassays were conducted at the University of Arizona Aquaculture Pathology Laboratory (UA-APL). Specific pathogen-free (SPF) whiteleg shrimp were obtained from a commercial supplier in Florida, USA. The SPF population has been screened for the past 2 years at UA-APL for all World Organization for Animal Health (OIE) listed and unlisted diseases, including EHP.
The EHP isolate used in this study was obtained from a population of whiteleg shrimp originating from Thailand. Two independent EHP trials were conducted. In both trials, shrimp were cultured at three different salinities of 2 ppt, 15 ppt, and 30 ppt. For each experimental challenge, six 90-liter tanks were filled with artificial seawater corresponding to the three salinity levels with two replicates for each salinity treatment. Temperature was regulated at 25 °C (± 0.6) with measurements taken every morning; pH was measured once a week with a range of 7.5–8.0.
Salinity was adjusted by changing 3 parts of salinity per hour from 25 ppt (initial salinity of SPF whiteleg shrimp population) to 5 ppt. To achieve salinity from 5 ppt to 2 ppt, the salinity was changed every 2 hours. Once established, salinity was measured with a refractometer once per week throughout the experimental period. Ten SPF whiteleg shrimp (2.0–2.1 g) were stocked in each tank for experimental infection. Three 90-l control tanks were set up for each salinity as negative controls.
Results and discussion
We investigated the prevalence and severity of EHP at three salinities: high (30 ppt), medium (15 ppt), and low (2 ppt) under laboratory challenge conditions. Fecal sequences used as the source of EHP in experimental challenges were sufficient to cause disease in shrimp cultured at different salinities, as confirmed by histopathology and PCR. The results from this study provide a novel method of EHP transmission via the fecal-oral route.
Final survival at the end of the EHP challenge was high, ranging from 90–100%, at 3 salinities in each of 2 independent challenge trials ( Table 1 ). Survival in the control treatments was 100% at all salinities in 2 independent challenge trials. We did not observe any clinical signs in shrimp exposed to EHP at the 3 different salinities.
Table 1. Final survival rates in shrimp in two independent challenge trials at three different salinities. Data are expressed as mean ± SD
Fecal strings were collected daily from pools known to be contaminated with EHP. Daily fecal string samples tested positive for EHP in both challenges. The mean weight of fecal strings added to each pool was 1.17 ± 0.52 grams and 0.32 ± 0.24 grams for challenges 1 and 2, respectively. The EHP copy number in the fecal strings used as inoculum was significantly higher in challenge 2 (p < 0.05). The EHP copy number in the fecal strings in challenge 1 was 1.6 × 10 3 ± 2.1 × 10 3 copies/ng DNA compared to 1.1 × 10 6 ± 2.0 × 10 6 copies/ng DNA in the challenge 2 inoculum.
The incidence and severity of EHP in the shrimp we tested using fecal pellets were assessed by H&E histology. In both trials, fecal pellets were able to cause disease in SPF shrimp. The incidence of EHP was 28.5% in trial 1 compared to 50% in trial 2. The data confirmed that fecal pellets were the source of EHP contamination in the trial.
The EHP infection rates at 2 ppt, 15 ppt, and 30 ppt salinities in challenge 1 were 25, 33.3, and 25%, respectively. In challenge 2, the EHP infection rates were 33.3, 30.0, and 87.5%, respectively. The severity was higher at 30 ppt salinity in challenge 2. In challenge 2, 50% of the EHP-infected population at 30 ppt salinity exhibited G3 (moderate to severe) and G4 (severe) lesions due to EHP infection. We found a strong correlation between salinity and EHP infection in shrimp, and the EHP incidence was higher in shrimp exposed to high salinity (30 ppt) than in shrimp exposed to low salinity (2 ppt and 15 ppt combined). The different severity levels in this study are shown in Figure 1.
Figure 1. H &E (Mayer-Bennet hematoxylin and eosin-phloxine) staining of shrimp hepatopancreas tissue showing the presence of different stages of EHP infection ac: level 0; df: level 1; gi: level 2; jl: level 3; mo: level 4 of EHP infection. Mature spores are indicated by blue stars. Black squares show typical regular-irregular plasmodium stages. Black circles show areas with EHP. Red squares outline magnified areas. Scale bars are located in the lower right part of each figure.
Figure 1, panels A, B and C show HP tissue sections at low, medium and high magnification of healthy shrimp from the control tank which did not show any histological lesions of EHP or any other pathogen. In contrast, panels D, E and F show HP tissue sections showing G1 level of EHP infection. A focal area within the HP was observed (Figure 1d). The affected hepatopancreatic tubules showed distinctive cytoplasmic inclusions in the cytoplasm of the affected tubular epithelial cells corresponding to the mononuclear meront stage (Figure 1e-f). Figure 1, panels G, H and I show G2 level of EHP infection (low to medium). The presence of EHP infection in some of the affected HP tubular epithelial cells was observed. Both the meront stage and the released spores were observed (Figure 1i). Figure 1, panels J, K, and L show typical G3 grade of EHP infection.
Multifocal lesions in hepatopancreatic tubular epithelial cells were observed (Figure 1j). In affected tubules, the presence of both irregular multinucleated plasmodium and spores in the cytoplasm of epidermal epithelial cells was observed (Figure 1). Figure 1 panels M, N, and O show G4 level of EHP infection with multifocal tubules containing infected HP cells (Figure 1m). Both multinucleated plasmodium and spores in the cytoplasm of affected cells as well as spores in the tubular lumen were observed (Figure 1o).
The hepatopancreas of SPF shrimp challenged with fecal sequences from EHP-infected shrimp tested positive for EHP using nested PCR in all tanks at three different salinities. This confirmed the presence of EHP in the treatment tanks for challenge 1 and challenge 2. EHP was detected at all three salinities evaluated (2 ppt, 15 ppt, and 30 ppt). Hepatopancreas tissue collected from negative control animals grown at 2 ppt, 15 ppt, and 30 ppt tested negative for EHP using nested PCR.
Previous studies have shown several routes of infection including cohabitation and direct inoculation into the hepatopancreas with EHP-infected material. In this study, we report for the first time that feces may be a route of infection. Due to detritus feeding behavior and the presence of undigested food in feces that can account for approximately 25–30%, shrimp in the treatment tanks ingested this food source along with EHP-infected spores, simulating the horizontal transmission observed at the farm level.
EHP, including the presence of the parasite in the cytoplasm of infected cells, and we found mature spores in the cytoplasm or released spores in the hepatopancreas by histopathology in EHP-challenged shrimp cultured at different salinities. This strongly suggests that EHP can cause infection at a wide range of salinities, ranging from 2 to 30 ppt. When the initial inoculum used for the experimental challenge was low (i.e. 1 × 10 3 copies of EHP/ng total faecal DNA), the incidence of HPM was similar (i.e. 25%) regardless of salinity.
However, EHP infection rates increased at 30 ppt salinity (87.5%), compared to 15 ppt (30%) and 2 ppt salinities (33.3%), when the challenge level increased from 1 × 10 3 to 1 × 10 6 EHP copies/ng total fecal DNA in the challenge experiment (challenge 2). Dose-dependent challenge has been well documented for other shrimp pathogens such as AHPND and Hepatobacter penaei. In the present study, the low copy number challenge strain of 1.6 × 10 3 used in challenge 1 caused mild infections in challenged shrimp. However, severe infections (G3 to G4 levels) and higher disease incidence occurred in challenge 2 when the EHP copy number was higher than 1.1 × 10 6 .
Histological lesions in shrimp maintained at 30 ppt salinity were more severe. Moderate to severe infection levels (G3-G4) were found in 50% of affected shrimp. In contrast, only 16% of shrimp maintained at 2 ppt salinity showed G3 infection levels and 0% of shrimp maintained at 15 ppt salinity showed G2-G4 infection levels. The difference in severity of EHP infection at the three different salinities may be due to the different effects of salinity on spore germination. One of the critical stages in spore germination is the increase in osmotic pressure within the spore. The difference in salinity results in a hypotonic (lower concentration) environment at 2 ppt and 15 ppt compared to a hypertonic (higher concentration) environment at 30 ppt. It is possible that hypertonic solutions promote spore germination by increasing spore activation.
Hardness was another variable that differed at the three salinities used in this study and may be a factor influencing spore germination. Hardness at low salinity (2 ppt) was approximately 240 mg/L compared to artificially generated seawater at 15 ppt and 30 ppt salinities of approximately 787 and 1575 mg/L, respectively. It has been reported that calcium is an important second messenger that triggers many cellular events and calcium influx may, in part, be responsible for triggering microspore release at higher salinities.
In commercial ponds in some EHP endemic areas in Asia, salinity conditions have been found to vary widely. For example, in India there are several high and low salinity shrimp farming areas, and the incidence of EHP appears to be lower at lower salinities (below 5 ppt), as observed in a shrimp disease survey in Andhra Pradesh in 2019. Similar conditions have been observed in two major shrimp farming areas in Venezuela, i.e. Lake Maracaibo, where salinities are around 4–6 ppt, and in Falcon State, where salinities range from 36 to 40 ppt. In Venezuela, shrimp farming is not fully integrated , and movement of nauplii and postlarvae between Falcon and Lake Maracaibo areas is a common practice. This suggests that EHP-infected PL and broodstock have been moved between these two areas. However, EHP has only been detected in the Falcon area, where salinity is high. In Lake Maracaibo, where salinity is low, EHP has not been reported. One possibility that limits the spread of EHP may be differences in water salinity.
Conclude
Our results demonstrate that fecal sequences from EHP-infected shrimp can be used as a reliable source of infection for experimental EHP infections via the fecal-oral route. EHP infection can occur at low salinities (i.e. 2 ppt) although disease incidence and severity are higher at 30 ppt salinity. These findings have implications for disease management in EHP-endemic areas.
Source: globalseafood
Translation: Marketing Department Than Vuong
