The final survival at the end of the EHP challenge was high, ranging from 90-100%, in the three salinities in each of the two independent experimental challenges (Table 1). The survival percentage in the control treatments were 100% in all of the salinities in the two independent challenge experiments. We did not observe any clinical signs in the shrimp exposed to EHP in the 3 different salinities.
Quantification of EHP in fecal strings by qPCR
Fecal strings were collected on a daily basis from known EHP-infected tanks. The daily fecal string samples tested positive for EHP in both challenges. The average weight of the fecal strings added to each tank was 1.17± 0.52 g and 0.32± 0.24 g for challenges #1 and #2, respectively.
The EHP copy number in the fecal strings used as inoculum was significantly higher in the challenge #2 (p<0.05). EHP copy number in the fecal string in challenge #1 was 1.6x103 ± 2.1x103 copies /ng DNA compared to 1.1x106 ± 2.0x106 copies /ng DNA in the inoculum of challenge #2 (Fig 1).
Prevalence of EHP by histopathology
The prevalence and severity of EHP in shrimp experimentally challenged using fecal strings as inocula in the two independent challenges was assessed by H&E histology.
In both experimental challenges, the fecal strings used as inocula were able to provoke the disease in SPF shrimp. The prevalence of EHP was 28.5% in the challenge #1 vs. 50.0% in the challenge #2. The data confirms that fecal strings are a reliable source of inoculum for experimental infection of EHP.
The prevalence of EHP at 2 ppt, 15 ppt, and 30 ppt salinities in challenge #1 was 25%, 33.3%, and 25%, respectively. In challenge #2, the prevalence of EHP at 2 ppt, 15 ppt, and 30 ppt was 33.3%, 30.0%, and 87.5%, respectively. The degree of severity was higher at a salinity of 30 ppt in the second experiment (Table 2). In challenge #2, 50% of the EHP-infected population at 30 ppt displayed grade G3 (moderate to severe) and G4 (severe) lesions caused by the EHP infection. A strong association between salinities and E. hepatopenaei-infected shrimp was found with an Odds Ratio (OR) of 4.3 (p =0.037). The prevalence of EHP in shrimp exposed to high salinity (30 ppt) was higher than shrimp exposed to low salinities (2 ppt and 15 ppt combined). The different grades of severity in this study are shown in Fig. 2. The Fig. 2 panels A, B, and C show tissue sections of HP at a low, medium, and high magnification of healthy shrimp from the control tank without showing any histological lesions of EHP or any other known pathogens. In contrast, panels D, E, and F show HP tissue sections displaying grade G1 of an EHP infection. A focalized region within the HP was observed (Fig. 2D). The affected tubule shows the distinctive cytoplasmic inclusion bodies in the cytoplasm of the affected epithelial tubule cells that correspond to the uninucleate meront stage (Fig 2E -F). Fig. 2 panels G, H, and I show grade G2 of an EHP infection (low to moderate). The focal presence of an EHP infection in few affected HP tubules epithelial cells were observed. Both, the meront stage and spores liberated into the lumen were observed (Fig 2I). Fig. 2 panels J, K, and L show a typical grade G3 of an EHP infection. Multifocal lesions in HP tubules epithelial cells were observed (Fig. 2J). In the affected tubules, the presence of both irregular multinucleated plasmodium and spores within the cytoplasm of the cuticular epithelial cells were observed (Fig. 2L). Fig. 2 panels M, N, and O show the grade G4 of an EHP infection with multifocal tubules containing infected HP cells (Fig 2M). Both multinucleated plasmodium and spores within the cytoplasm of the affected cells as well as spores within the lumina of the tubules were observed (Fig 2O).
Detection of EHP in hepatopancreas by PCR
The hepatopancreas of SPF shrimp challenged with fecal strings obtained from EHP-infected shrimp had positive results for EHP using nested PCR in all of the tanks at the three different salinities. This confirms the presence of EHP in the treatment tanks for challenge #1 and challenge #2. In all three (3) salinities (2 ppt, 15 ppt, and 30 ppt) EHP was detected. The hepatopancreas tissue collected from the negative control treatment animals reared at the 2 ppt, 15 ppt, and 30 ppt salinities tested negative for EHP using nested PCR.