Compositions and related methods for inhibiting EHP infection in shrimps

Abstract

The present invention relates to methods of applying a composition comprising thymoquinone in an amount effective to inactivate EHP spores and to reduce the rate of EHP infection wherein thymoquinone may be derived from Mentha America, Mentha pseudosciaena, or fennelflower foeniculata. The invention also relates to a method for controlling the proliferation of EHP in aquaculture, in particular the propagation of EHP infection in a population of shrimps. Another aspect of the invention relates to a method for controlling intracellular proliferation of EHP by directly inactivating EHP spores by administering to shrimps a composition containing thymoquinone, such as a composition containing nigella foeniculata seed oil. Another aspect of the invention relates to compositions containing only nigella foeniculata seed extract or in oil, in at least one embodiment, in combination with American mint oil, origanum oil, clove oil and cinnamyl aldehyde to control and inhibit the propagation of EHP infection and its related diseases.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of Indian Provisional Patent Application No. 202211065837, filed on November 17, 2023, entitled "COMPOSITIONS FOR INHIBITING EHP INFECTION IN SHRIMP AND RELATED METHODS", the entire disclosure of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Hepatopancreatic microsporidiosis (HPM) is a microsporidian infection in shrimp caused by the intracellular microsporidian parasite Enterocytozoon hepatopenaei (EHP). The shrimp farming industry has suffered significant economic losses due to EHP infection due to limited methods for detecting the infection and a lack of methods for controlling the spread of EHP. Thawatchai, C. et al. (2021), The shrimp microsporidian Enterocytozoon hepatopenaei (EHP): Biology, pathology, diagnostics and control, Journal of Invertebrate Pathology, 186:107458; Thitamadee S. et al., (2016), Review of current disease threats for cultivated penaeid shrimp in Asia, Aquaculture, 452:69–87; Chao Ma et al., (2021), Rapid detection of Enterocytozoon hepatopenaei infection in shrimp with a Real-Time isothermal recombinase polymerase amplification assay, Frontiers in Cellular and Infection Microbiology, Vol. 11, 1-8.

[0004] The shrimp hepatopancreas (HP) is a central organ that secretes digestive enzymes, absorbs and stores nutrients. EHP is an emerging pathogen that primarily infects the tubular epithelial cells of HP and causes severe growth retardation and size variability in shrimp. EHP is also associated with other bacterial diseases, among which shrimp infected with EHP have been found to be highly susceptible to infection with various Vibrio species, resulting in massive mortality. See Han, JL et al. (2019), Molecular detection of Enterocytozoonhepatopenaei and Vibrio parahaemolyticus-associated acute hepatopancreaticnecrosis disease in Southeast Asian Penaeus vannamei shrimp imported into Korea, Aquaculture, 517: 734812. Currently, EHP is a limiting factor to the sustainability of global shrimp production because the disease is widely spread in shrimp farming countries without an effective solution to control the spread. Summary of the invention

[0005] The present invention relates to compositions for controlling the proliferation of EHP in aquaculture, in particular compositions capable of controlling the spread of EHP in shrimp populations. One aspect of the present invention relates to compositions containing N. sativa seed extract / oil alone or in combination with essential oils (monarda oil, oregano oil, clove oil and cinnamaldehyde) that can control and inhibit the spread of EHP infection and its related diseases. According to at least one embodiment, the composition of the present invention is administered to shrimp as a feed additive.

[0006] Another aspect of the invention relates to methods of causing direct inactivation of EHP spores to prevent or reduce infection in shrimp. Another aspect of the invention relates to administering a composition, such as an animal feed additive included in an animal feed, in an amount effective to control the spread of EHP. Another aspect of the invention relates to orally administering a composition to shrimp in an amount effective to inhibit the proliferation of EHP in a shrimp population, and administering the composition to shrimp in an amount effective to control HPM.

[0007] The present inventors have discovered compositions capable of inactivating the EHP polar tube extrusion mechanism (adhesion mechanism), a development that has important implications for controlling the first step of infection, namely the adhesion mechanism of EHP sporoplasm translocation into host cells.

[0008] In addition, the present inventors have found a composition capable of inactivating the EHP post-translational modification enzyme methionine aminopeptidase 2 (MetAP2), which has the prospect of controlling the increase in the intracellular level of EHP. The present inventors have surprisingly found a composition that effectively inhibits the proliferation of EHP and is also considered safe for oral administration to shrimp, for example, through supplements in feed.

[0009] Detailed Description of the Drawings

[0010] Figure 1 Depicts the effect of bioactive molecules on polar tube extrusion of batch 1 spores. All data are represented as mean ± SD, N = 3. Asterisks show significant differences in extrusion % compared to the dimethyl sulfoxide (DMSO) control (p = 0.002).

[0011] Figure 2 Depicts the effect of bioactive molecules on polar tube extrusion of batch 2 spores. All data are represented as mean ± SD, N = 3. Asterisks show significant differences in extrusion % compared to the DMSO control (p = 0.002).

[0012] Figure 3A and 3B are micrographs of -EHP spores with polar tube extrusion under microscopy (100X). Representative images of spores from batch 2 treated with 100 ppm thymoquinone (BAM1) and control.

[0013] Figure 4 Depicts the total blood cell count at different time intervals. Data are represented as mean ± SD: N = 3 (p < 0.05).

[0014] Figure 5 Depicts the aspartate aminotransferase (AST) activity at different time intervals. Data are represented as mean ± SD: N = 3 (p < 0.05).

[0015] Figure 6 Depicts the alkaline phosphatase (AP) activity at different time intervals. Data are represented as mean ± SD: N = 3 (p < 0.05).

[0016] Figure 7 Depicts the total blood cell count at different time intervals. Data are represented as mean ± SD: N = 3 (p < 0.05).

[0017] Figure 8 Depicts the prophenoloxidase (ProPO) activity at different time intervals. Data are represented as mean ± SD: N = 3 (p < 0.05).

[0018] Figure 9Depicts the superoxide dismutase (SOD) activity at different time intervals. Data are presented as mean ± SD: N = 3 (p < 0.05).

[0019] Figure 10 Describes the relative penaeidin-3a gene expression at different time intervals. Data are presented as mean ± SD: N = 3 (p < 0.05).

[0020] Figure 11 Describes the relative crustin gene expression at different time intervals. Data are presented as mean ± SD: N = 3 (p < 0.05).

[0021] Figure 12 Depicts the relative anti-lipopolysaccharide (ALF) factor gene expression at different time intervals. Data are presented as mean ± SD: N = 3 (p < 0.05).

[0022] Figure 13 Depicts the relative Toll-like receptor (TLR) gene expression at different time intervals. Data are presented as mean ± SD: N = 3 (p < 0.05).

[0023] Figure 14 Depicts the alanine aminotransferase (ALT) activity at different time intervals. Data are presented as mean ± SD: N = 3 (p < 0.05).

[0024] Figure 15 Depicts the aspartate aminotransferase (AST) activity at different time intervals. Data are presented as mean ± SD: N = 3 (p < 0.05).

[0025] Figure 16 Depicts the alkaline phosphatase (AP) activity at different time intervals. Data are presented as mean ± SD: N = 3 (p < 0.05).

[0026] Figure 17 Depicts the survival rate (%) of shrimp after 120 h of bacterial challenge with Vibrio parahaemolyticus at 2.6 × 10 3 CFU / ml. All values are presented as mean ± SD, where n = 4 (30 shrimps in each replicate) (P < 0.05).

[0027] Figure 18 depicts the cohabitation challenge model (Salachan et al., 2016). A – Model of infected control and treatment groups. B – Model of uninfected control group

[0028] Figure 19Depicts the weekly weight gain at different time intervals. All data are presented as mean ± SD, n = 20 (5 shrimps per replicate) (p < 0.05).

[0029] Figure 20 Depicts the final weight gain, feed conversion ratio, and specific growth rate of different experimental groups. Data are presented as mean ± SD; n = 4 (25 shrimps per replicate): (P < 0.05).

[0030] Figure 21 Depicts the final survival rate (%) of different experimental groups. Data are presented as mean ± SD; n = 4 (25 shrimps per replicate): (P < 0.05).

[0031] Figure 22 Depicts the quantification of EHP by qPCR. All values are presented as mean ± SD, where n = 4 (P < 0.05)

[0032] Figure 23 Depicts the final biomass production and feed conversion ratio of the experimental ponds.

[0033] Figure 24 Depicts the final survival rate and specific growth rate of the experimental ponds.

[0034] Figure 25 Depicts the final size change between the control ponds and the treatment ponds. Detailed implementation mode

[0035] Plant-derived components are potential sustainable sources of antimicrobial chemotypes and have not been exploited as potential solutions for controlling pathogens. The present inventors surprisingly found that dietary supplements of Nigella sativa extract / oil alone or in combination with essential oils such as Monarda didyma oil, Origanum vulgare oil, Syzygium aromaticum oil, and Cinnamomum cassia bark oil demonstrated a statistically significant reduction in EHP infection in shrimp. The present inventors also demonstrated that Aquavibra TM (Kemin Industries, Inc.), a combination of cinnamaldehyde and Origanum vulgare oil, was able to achieve a significant reduction in EHP infection.

[0036] Nigella sativa is an annual herbaceous plant belonging to the Ranunculaceae family. Nigella sativa seeds are rich in terpenoids, including thymoquinone, p-cymene, γ-terpinene, β-pinene, carvacrol, terpinen-4-ol, and longifolene.

[0037] The present invention relates to a composition comprising Nigella arvensis L. seed oil, for controlling the proliferation of EHP in aquaculture, particularly for controlling the proliferation of EHP in aquaculture in an amount capable of controlling the spread of EHP infection in shrimp populations. According to at least one embodiment of the present invention, the composition further comprises at least one essential oil, which includes but is not limited to those selected from the following: Nigella arvensis L. seed oil / extract, cinnamaldehyde, oregano oil, cinnamon bark oil, clove oil, Monarda didyma oil or combinations thereof. In a preferred embodiment, the composition contains Nigella arvensis L. seed oil in combination with oregano oil, Monarda didyma oil, clove oil and cinnamaldehyde. In an alternative embodiment, the composition of the present invention may comprise thymoquinone, thymohydroquinone, dithymoquinone, p-cymene, carvacrol, terpineol, thymol and combinations thereof.

[0038] In certain embodiments, the composition of the present invention contains from about 0.1% to 100% by weight, such as from 1% to 95% by weight, or from 5% to 95% by weight of Nigella arvensis L. seed oil. In certain embodiments, the composition further contains oregano oil in an amount of about 0 to 99.9% by weight, Monarda didyma oil in an amount of about 0 to 99.9% by weight, clove oil in an amount of about 0 to about 50% by weight and cinnamaldehyde in an amount of about 0 to about 50% by weight. In certain embodiments, the composition optionally comprises thymoquinone, thymohydroquinone, dithymoquinone, p-cymene, carvacrol, terpineol and / or thymol, wherein such optional components are present in an amount of about 0.1% to about 50% by weight.

[0039] Another aspect of the present invention relates to directly inactivating EHP spores in order to control the adhesion mechanism and intracellular proliferation by altering post-translational modifications. In some embodiments, oral administration of a composition containing an effective amount of Nigella arvensis L. seed oil results in a reduction of EHP infection by about 0 to 100%, such as a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in infection.

[0040] Another aspect of the present invention relates to a composition containing an effective amount of Nigella arvensis L. seed oil, which results in a reduction of EHP infection, such as as a supplement or additive to animal feed, wherein the composition is included in the animal feed in an amount effective to control the spread of EHP. According to at least one embodiment, the composition of the present invention is included in the feed in an amount of about 0.1 g / kg to 20 g / kg of feed, such as about 0.5 g / kg to 10 g / kg of feed.

[0041] Another aspect of the invention relates to applying the composition to the shrimp in an amount effective to inhibit EHP in the shrimp population and applying the composition to the shrimp in an amount effective to control HPM.

[0042] Another aspect of the invention relates to compositions capable of inactivating EHP infection involving extrusion of polar tubes (adhesion mechanism).For example, in some embodiments, the EHP infection rate in a shrimp population is reduced while EHP spores continue to proliferate.

[0043] Another aspect of the invention relates to a composition that is effective in inhibiting EHP proliferation and at the same time safe for administration to shrimp, such as through a supplement in animal feed. In an alternative embodiment, the composition can be added to a water source. In another alternative embodiment, the composition can be applied to water or soil sediments containing EHP spores.

[0044] Another aspect of the invention involves applying an amount of the composition effective to inactivate EHP spores present in shrimp culture water and shrimp pond soil sediments.

[0045] The following examples illustrate the invention and are not intended to be limiting.

[0046] Examples

[0047] Example 1

[0048] The selected bioactive compounds (carvacrol, α-terpineol, thymoquinone, cinnamaldehyde and eugenol) and positive controls (fumagillin and albendazole) were subjected to in silico molecular docking and molecular dynamics (MD) simulations with the microsporidian drug target methionine aminopeptidase 2 (MetAP2), as summarized in Table 1. The results revealed promising inhibitory potentials of three lead compounds: carvacrol, α-terpineol and thymoquinone against MetAP2, with binding energies comparable to those of the controls. Molecular mechanics with generalized Born and surface area solvation (MM / GBSA) and Poisson-Boltzmann surface area (MM / PBSA) calculations revealed thermodynamically stable binding energies of the compounds with MetAP2. The binding and inhibition of the compounds against MetAP2 were demonstrated to be thermodynamically favorable, as summarized in Tables 2 and 3.

[0049] Table 1. Binding affinities of bioactive molecules and positive controls to amino acids.

[0050]

[0051]

[0052]

[0053] Table 2: Thermodynamic parameters of the complexes of lead compounds and positive controls with 3FMQ (target) calculated by the MM-GBSA method.

[0054]

[0055] Table 3. Thermodynamic parameters of the complexes of lead compounds and positive controls with MetAP2 (3FMQ) calculated by the MM-PBSA method.

[0056]

[0057] All values are expressed as mean ± SD.

[0058] Example 2

[0059] The EHP infection mechanism involves the extrusion of polar tubes (adhesion mechanism). In in vitro studies, the inactivation of polar tube extrusion of EHP spores was tested with selected active ingredients, namely thymoquinone, cinnamaldehyde, eugenol, and carvacrol. Stock solutions of bioactive molecules were prepared in dimethyl sulfoxide (DMSO), and further working solutions were prepared with 1X sterile phosphate buffer saline (1XPBS).

[0060] EHP spores were isolated and purified from the HP of infected shrimp from a high-salt shrimp culture system (batch 1) and a low-salt shrimp culture system (batch 2). The purified spores were then incubated with the test molecules at 37 °C for 2 hours at the desired concentration. Subsequently, the washed spores were induced to extrude polar tubes with an alkaline reagent. The extrusion of spores was determined by counting the number of extruded spores from a total of 100 spores under an optical microscope at 100X magnification and converting this number into extrusion %.

[0061] In the results of batch 1 spores, thymoquinone at 10 ppm and 100 ppm showed significantly reduced polar tube extrusion rates of 43.92% and 6.34%, respectively, as Figure 4 shown. Similar results were replicated in the batch 2 spore study, and a dose-dependent effect of thymoquinone was observed in the results of both batch 1 and batch 2. Compared with 86.47% and 88.32% extrusion of the 2% and 20% DMSO controls, only 40.31% and 0.31% were found after incubation with 10 ppm and 100 ppm thymoquinone, respectively, as Figure 1 and 2Shown. Micrographs of EHP spores extruded from polar tubes in batch 1 and batch 2 studies are shown in Figure 3. The results clearly show that the composition containing thymol will be a promising candidate for controlling EHP infection in shrimp by its ability to inhibit polar tube extrusion. Compared with the DMSO control, other bioactive molecules such as cinnamaldehyde, carvacrol, and eugenol had a slight reduction in polar tube extrusion of EHP spores at a higher concentration of 100 ppm (p > 0.05).

[0062] Statistical analysis. One-way analysis of variance (ANOVA) and t-tests were performed to determine the significant differences in the percentage of polar tube extrusion between the spores treated with bioactive molecules and the corresponding DMSO controls at a 95% confidence interval. The analysis was performed using STATGRAPHICS centurion.

[0063] Example 3

[0064] In vivo study (40 days) was conducted in shrimp with uninfected and infected controls and two treatment groups. The treatment groups included: 0.5% Nigella sativa leaf extract (Sample 1), and a combination of cinnamaldehyde and oregano oil (Sample 2). In the experimental design, each group included 3 replicates, and each replicate included 15 shrimp. Water changes (20% - 30%) were completed every two days. The aquariums were maintained at the recommended temperature (28°C - 30°C) and dissolved oxygen (> 5 mg / L). The shrimp were fed the experimental diet 3 times a day at 3% to 3.5% of their body weight for 40 days.

[0065] Shrimp were screened for EHP and other shrimp pathogens by PCR testing methods. Animals that were EHP positive but free of other infections were selected as the infected and treatment groups. Age-matched shrimp that were negative for EHP and other infections were used as the uninfected controls.

[0066] Data collection. Data were collected considering: (1). The immune status of the shrimp. The total hemocyte count (THC) was determined by counting the cells in a droplet of anticoagulant-mixed hemolymph placed under a microscope. (2). Hepato-pancreatic health marker enzymes. Aspartate aminotransferase (AST) and alkaline phosphatase (AP) in shrimp hemolymph were determined by spectrophotometry. (3). Quantification of EHP. The shrimp hepatopancreas was aseptically dissected, DNA was extracted, and EHP infection was quantified by qPCR.

[0067] Results. The treatment groups showed significantly higher total hemocyte counts than the controls (p < 0.05), indicating an enhanced health status of the shrimp, as Figure 4 shown. Elevated levels of liver marker enzymes in the hemolymph indicated acute injury to the HP. Compared with the infected controls from day 20, the marker enzymes AST and AP were significantly (p < 0.05) reduced in the treatment, as Figure 5 and 6As shown. Compared with the infected control, the EHP DNA quantification by qPCR showed a gradual increase in the threshold cycle values in the treatment groups from day 0 to day 40. Sample-1 at 0.5% inclusion rate showed a significant increase in the threshold cycle values and a lower EHP DNA copy number (Table 4).

[0068] Table 4. EHP quantification - RT PCR in hepatopancreas

[0069]

[0070] Example 4

[0071] In vivo study (40 days) was conducted in shrimps with uninfected and infected controls and three treatment groups. The treatment groups included: 0.5% Nigella sativa oil (Sample 1) and a combination group (Sample 2) with two different inclusion rates (0.5% and 1% by weight in the feed). A combined sample containing Nigella sativa oil, oregano oil, clove oil, and cinnamaldehyde was prepared. In the experimental design, there were 4 replicates per group, and 40 shrimps were maintained per replicate. Water change (20% - 30%) was completed every two days. The aquariums were maintained at the recommended temperature (28°C - 30°C) and dissolved oxygen (>5 mg / L). The shrimps were fed the experimental diet 3 times a day at 3% - 3.5% of their body weight for 40 days. After the feeding trial, the animals were challenged with Vibrio parahaemolyticus at the LD 30 dose.

[0072] For the experiment, the shrimps were screened for EHP and other shrimp pathogens by PCR test method. Animals that were EHP positive but without other infections were selected as the infected and treatment groups. Age-matched shrimps that were negative for EHP and other infections were used as the uninfected control.

[0073] Data collection. Considerations for data collection include: (1) Shrimp immune status. The total hemocyte count (THC), prophenoloxidase activity (proPO), and superoxide dismutase (SOD) activity were evaluated at 10-day intervals. THC was determined by counting the cells in a droplet of anticoagulated mixed hemolymph placed under a microscope. ProPO activity was determined by spectrophotometry, measuring the formation of dopachrome from L-dihydroxyphenylalanine (L-dopa). SOD was evaluated by spectrophotometry based on the auto-oxidation of adrenaline. (2) Immune gene expression. The immune gene expression profiles of shrimp antimicrobial peptide 3a, crustin, anti-lipopolysaccharide factor, toll-like receptor, and β-actin (housekeeping gene) were studied by qRT-PCR. qRT-PCR was performed in triplicate. Relative quantification was evaluated using the Ct value. For each sample, the Ct value of the target gene was subtracted from the value of the housekeeping gene (β-actin) to normalize the relative expression level of the gene to obtain the ΔCt value. The ΔCt value of the test samples (infected control and treatment groups) was subtracted from the ΔCt value of the calibration sample (uninfected control) to obtain the ΔΔCt value. (3) Hepatopancreas health marker enzymes. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AP) in shrimp hemolymph were determined by spectrophotometry. (4) Quantification of EHP. The shrimp hepatopancreas was aseptically dissected, DNA was extracted, and EHP was quantified by qPCR. (5) Experimental immersion challenge with Vibrio parahaemolyticus. After the feeding study, the shrimp were transferred to challenge tanks (100 L capacity and 60 L water), and similar salinity, aeration, and corresponding feed were followed. In each tank, 30 shrimp were stocked and acclimated for 40 h. A field isolate of Vibrio parahaemolyticus was used for the immersion challenge study. After completion of the acclimation period, the shrimp were immersed in a Vibrio parahaemolyticus suspension at the LC30 dose (2.6×10 3 CFU / mL), while the bacterial suspension in the uninfected group was replaced with sterilized seawater. The mortality of the shrimp was recorded every 6 h until 5 days after immersion.

[0074] Results. Compared with the treatment groups on day 20, the total hemocyte count showed a significant decrease in the infected control as the disease progressed. As Figure 7 shown, the treatment groups showed an initial decrease until day 20 and an enhanced THC level in the following days. Compared with the infected control, all treatment groups showed a significant (p<0.05) increase in THC from day 20. All treatment groups showed a gradual decrease in the ProPO and SOD activity levels from day 1 to day 10, as Figure 8 and 9 shown. Compared with the uninfected control and treatment groups, the infected control showed significantly higher levels of ProPO and SOD activity from day 10 to day 40.

[0075] Immune gene expression. Compared with the initial level, the expression of shrimp antimicrobial peptide-3a decreased in all treatment groups. However, as Figure 10 shown, on day 40, the infected control showed a higher expression of shrimp antimicrobial peptide-3a compared with the initial level and a significantly higher expression compared with other treatment groups. The expression of crustin varied at different time intervals. On day 40, all treatment groups showed a lower level of crustin expression compared with the infected control. In the treatment groups, the combined group (sample 2) showed a gradually decreasing level of crustin expression, as Figure 11 shown. The expression of ALF and TLR in sample 1 was significantly (p < 0.05) higher from day 20 and day 30, respectively, compared with the infected control and other treatment groups. On day 40, the ALF level in the combined group (sample 2) was significantly lower compared with other groups, as Figure 12 shown. The TLR expression in sample 1 was significantly higher from day 30 compared with other groups, as Figure 13 shown.

[0076] Hepatopancreas health marker enzymes. The levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase in the hemolymph of the infected and treatment groups were higher on day 1 compared with the uninfected group. After day 10, the levels in all treatment groups were significantly (p < 0.05) lower compared with the infected control. The infected control had a high level of marker enzymes throughout the study period. The AST and ALT levels in the combined group (sample 2) were significantly lower from day 10 compared with the infected control and other treatment groups, as Figure 14 and 15 shown. On day 40, the AP level in the combined group was significantly (p < 0.05) lower compared with the infected control and other treatment groups, as Figure 16 shown.

[0077] Quantification of EHP. The treatment groups showed a gradual increase in the C T value compared with the infected control. All treatment groups showed a lower copy number on day 40 compared with the infected control (Table 5). In the treatment, sample 2 (the combined group with 5 g / kg feed) showed a significantly lower copy number (35) compared with other groups.

[0078] Table 5. EHP quantification in the hepatopancreas - RT PCR (Data are presented as mean ± SD: N = 3 (p < 0.05))

[0079]

[0080] Experimental immersion challenge with Vibrio parahaemolyticus. The infected control group showed a significantly lower final survival rate compared with the uninfected control and treatment groups, as Figure 17As shown. There were no significant differences between the treatment groups. However, compared to the other treatment groups, the combination group (Sample 2) showed improved survival.

[0081] The researchers observed an improvement in the immune status in the treatment groups. Hemocytes are the blood cells of invertebrates that are involved in key activities of innate immunity. The treatment groups showed improved hemocyte counts after an initial decline, representing an increase in disease resistance. The prophenoloxidase (proPO) cascade is an important innate immune response against microbial infection in invertebrates. The antioxidant enzyme superoxide dismutase (SOD) converts this microbicidal metabolite into hydrogen peroxide that freely passes through the membrane. The maintenance of the initial levels of proPO and SOD activities in the infected controls and the gradually decreasing activities in the treatment groups indicate recovery from the infection.

[0082] Antimicrobial peptides (AMPs) are part of an important first-line response in the host defense system. The cationic AMPs in penaeid shrimp, which consist of penaeidin, crustin, and anti-lipopolysaccharide factor, are composed of multiple classes or isoforms and have antibacterial and antifungal activities against different strains of bacteria, fungi, and enveloped viruses. Penaeidin, which acts mainly against Gram-positive bacteria and filamentous fungi, consists of an N-terminal domain rich in proline residues and a C-terminal domain containing six cysteines that form three disulfide bonds. The expression of penaeidin-3a gradually decreased from the initial level in all treatment groups, indicating a reduction in infection. Similarly, the reduced expression of ALF and crustin in the combination group confirmed lower bacterial and fungal infections in the shrimp. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single-pass transmembrane non-catalytic receptors that recognize structurally conserved molecules derived from microorganisms. The high relative expression of TLRs in the groups of Nigella arvensis alone and in combination demonstrated the immunomodulatory effects of the bioactive compounds present in Nigella arvensis seeds / oil and their combination with the essential oil.

[0083] The treatment group showed significantly reduced hepatopancreas health marker enzymes, such as AST, ALT, and AP, which indicated the recovery of hepatopancreatic epithelial cells from the damage caused by EHP infection. Previous studies supported that elevated levels of AST and ALT in hemolymph were associated with necrosis and extensive damage in hepatopancreatic cells due to severe EHP infection and were observed in naturally and experimentally EHP-infected shrimp. Santhoshkumar, S. et al., (2017), Biochemical changes and tissue distribution of Enterocytozoon hepatopenaei (EHP) in naturally and experimentally EHP-infected white leg shrimp, Litopenaeus vannamei, J. Fish Dis. 40(4):529-539.

[0084] The production of lower HP health marker enzymes was consistent with the EHP DNA quantification results. Compared with the infected controls, the qPCR results showed a gradual increase in the threshold cycle values in the treatment group from day 0 to day 40. The combined group (sample 2) significantly (p<0.05) reduced the EHP DNA copy number, and it should be understood that EHP-infected shrimp have a higher susceptibility to Vibrio-related diseases. Luis, F.A. et al., (2017), Enterocytozoon hepatopenaei (EHP) is a risk factor for acute hepatopancreatic necrosis disease (AHPND) and septic hepatopancreatic necrosis (SHPN) in the Pacific white shrimp Penaeus vannamei, Aquaculture, 471, 37-42. Higher survival was observed in the treatment group against Vibrio parahaemolyticus challenge, indicating a lower susceptibility to Vibrio infection.

[0085] Example 5

[0086] Mature EHP spores are infectious and prevalent in the shrimp farming system from hatcheries to grow-out ponds. Therefore, healthy shrimp have a high probability of being exposed to and infected by mature spores. The samples were mixtures of plant extracts tested in a cohabitation challenge model to evaluate the effects of the supplements in an environment of continuous and substantial exposure to mature EHP spores excreted by infected shrimp.

[0087] In vivo experiments (40 days) were conducted to study the effect of supplementing samples (a combination of Nigella sativa oil, Monarda fistulosa oil, Oregano vulgare oil, Syzygium aromaticum (clove) oil, and cinnamaldehyde) on resistance to Enterocytozoon hepatopenaei in Litopenaeus vannamei through cohabitation challenge. The experimental design consisted of four groups, including two controls (uninfected control and infected control), and the samples were top-dressed on the feed at doses of 0.3% and 0.5%, with 8 replicates each. In each replicate of the infected control and sample groups, EHP-infected shrimp were reared in cages, while healthy shrimp were placed outside the cages in the same tank ( Figure 18A ). A similar setup was followed for the uninfected group, but with healthy shrimp both inside and outside the cages ( Figure 18B ). The details of the experimental groups are mentioned in Table 6. Growth parameters and EHP infection rates were measured at different time intervals. All tanks were maintained at a recommended temperature range of 28°C - 30°C and a dissolved oxygen level of more than 5 mg / L. The shrimp were fed the experimental diet three times a day at 3% - 3.5% of their body weight for a period of 40 days.

[0088] Table 6. Details of the experimental groups.

[0089]

[0090] Selection of experimental animals. The selection criteria for infected animals were that they were positive for EHP testing and negative for other shrimp pathogens. Animals that tested negative for all disease tests were selected as healthy shrimp.

[0091] Data collection. (1) Growth performance parameters were recorded, including weekly weight gain, feed conversion ratio (FCR), specific growth rate (SGR), and survival rate of shrimp outside the cages. (2) Quantification of EHP – The hepatopancreas of shrimp was dissected separately and aseptically from each representative shrimp sample outside the cages. One shrimp was taken from each replicate, and DNA was extracted for EHP quantification using qPCR.

[0092] Statistical analysis. The statistical differences at a significant level (p-value < 0.05) between treatments were calculated by one-way ANOVA using STATGRAPHICS centurion software.

[0093] Results. The researchers observed that at all time intervals, the weekly weight gain in the sample-supplemented groups was significantly higher (p < 0.05) compared to the infected control group ( Figure 19)。By day 40, the final weight gain of the 0.5% sample group (11 ± 1.96 g) was comparable to that of the uninfected control group (12.75 ± 1.57 g). Additionally, compared to the infected control group, the sample supplement resulted in a reduced FCR and increased SGR%, as Figure 20 shown. The survival rate in the sample-supplemented group (>80%) was significantly improved (p < 0.05) compared to the infected control group (70%) ( Figure 21 ).

[0094] The infected control group showed significantly higher Enterocytozoon hepatopenaei (EHP) DNA copy numbers (8650 / μL) during the 5-day period after challenge and maintained this higher infection level (with higher EHP DNA copies) in the infected control group. In contrast, compared to the infected control group, the sample-supplemented group showed negative PCR results 5 days after challenge and maintained significantly lower EHP copies throughout the 40-day challenge period. By day 40, the group supplemented with 0.5% sample showed a higher fold reduction in EHP copies (67-fold) compared to the group supplemented with 0.3% sample (12-fold), as Figure 22 shown. This confirmed that the sample supplement delays EHP infection and significantly reduces the infection rate under severe challenge conditions.

[0095] The results indicate that the sample (a composition containing Nigella sativa oil, Monarda fistulosa oil, Syzygium aromaticum oil, Origanum vulgare oil, and cinnamaldehyde) in this study can be a functional ingredient in shrimp feed to increase disease resistance against EHP infection and serve as a protective tool against shrimp production losses caused by EHP infection.

[0096] Example 6

[0097] The researchers studied the effect of a sample (a combination of Nigella sativa oil, Monarda fistulosa oil, Syzygium aromaticum oil, Origanum vulgare oil, and cinnamaldehyde) supplement on the resistance of Litopenaeus vannamei reared in earthen shrimp culture systems against Enterocytozoon hepatopenaei. The initial EHP infection rate was confirmed by qPCR. The sample surface was added to commercial feed using a suitable binder and administered to the infected shrimp at inclusion rates of 0.5% and 1% in their diet for a duration of 30 days. The infection rate was determined at regular time intervals. After the initial 30-day period, the sample supplement was stopped and the shrimp were fed a control diet for 20 days to assess the reinfection rate. This was done by quantifying the EHP DNA copies in the hepatopancreas of the shrimp using qPCR. Additionally, growth parameters including biomass production, FCR, SGR, and survival were recorded and compared to an adjacent shrimp culture system without EHP infection (control).

[0098] Table 7. Quantification of EHP infection by qPCR.

[0099]

[0100] The present inventors observed a significant reduction in EHP infection in the sample replenishment pond, as described in Table 7. Growth, survival, and feed efficiency were improved using the sample supplement compared to the control ponds shown in Figure 23 and 24 . Additionally, as shown in Figure 25 , there was a reduced change in size in the sample-supplemented shrimp.

[0101] As described throughout the present disclosure, the inventors surprisingly found that Nigella sativa seed extract / oil alone or in combination with one or more essential oils results in a synergistic effect in controlling and inhibiting the spread of EHP infection and its associated diseases, the essential oils including but not limited to oregano oil, Monarda oil, clove oil, and cinnamaldehyde.

[0102] In at least one embodiment, the present inventors surprisingly found a method for controlling the spread of EHP infection and related diseases in shrimp, the method comprising orally administering to the shrimp a composition comprising an amount of thymoquinone effective to reduce the EHP infection rate. In certain embodiments, thymoquinone is present in an amount of at least 100 ppm, such as 200 ppm, 300 ppm, 400 ppm, or 500 ppm. In certain embodiments, thymoquinone is derived from Monarda didyma, Monarda fistulosa, or Nigella sativa.

[0103] In at least one embodiment, the composition of the present invention further comprises one or more essential oils selected from Monarda oil, oregano oil, clove oil, and cinnamaldehyde. As a non-limiting example, in at least one embodiment, the composition of the present invention comprises from about 0.01% to about 100% by weight of thymoquinone, from about 0 to about 70% by weight of carvacrol, from about 0 to about 70% by weight of α-terpineol, from about 0 to about 30% by weight of cinnamaldehyde, and from 0 to about 30% by weight of eugenol.

[0104] In at least one embodiment, the composition of the present invention is administered by adding the composition to the animal diet, such as adding the composition to the diet of shrimp as an animal feed additive. For example, the composition is added to the feed in an amount of at least about 0.1% by weight of the feed, and in certain embodiments in an amount of about 0.1% to about 2% by weight of the feed.

[0105] In an alternative embodiment, the composition is added to water or a pond, wherein the composition is administered in an amount effective to inactivate EHP spores present in shrimp aquaculture water and shrimp pond soil sediment.

[0106] The present inventors have also discovered a method for inactivating EHP spores, the method comprising oral administration to shrimp of a composition comprising thymquinone, such as a composition comprising Nigella sativa seed extract / oil and one or more essential oils / extracts, in an amount effective to inactivate EHP spores and reduce the EHP infection rate. In certain embodiments, the compositions of the present invention further comprise one or more essential oils, including but not limited to Monarda didyma oil, Origanum vulgare oil, Syzygium aromaticum oil, and cinnamaldehyde / cinnamon oil. In at least one embodiment, the composition of the present invention comprises thymquinone in an amount of about 0.01% to about 100% by weight, carvacrol in an amount of about 0 to about 70% by weight, α-terpineol in an amount of about 0 to about 70% by weight, cinnamaldehyde in an amount of about 0 to about 30% by weight, and eugenol in an amount of 0 to about 30% by weight.

[0107] Another embodiment of the present invention relates to a feed additive composition comprising thymquinone in an amount effective to inactivate EHP spores and reduce the EHP infection rate. In certain embodiments, the thymquinone is derived from Monarda didyma, Monarda fistulosa, or Nigella sativa. In certain embodiments, the feed additive contains Nigella sativa extract or oil. In at least one embodiment, the feed additive further comprises one or more essential oils, including but not limited to Monarda didyma oil, Origanum vulgare oil, Syzygium aromaticum oil, and cinnamaldehyde / cinnamon oil. For example, in at least one embodiment, the composition of the present invention comprises thymquinone in an amount of about 0.01% to about 100% by weight, carvacrol in an amount of about 0 to about 70% by weight, α-terpineol in an amount of about 0 to about 70% by weight, cinnamaldehyde in an amount of about 0 to about 30% by weight, and eugenol in an amount of 0 to about 30% by weight.

[0108] In certain embodiments, the feed additive is added to the shrimp diet as an animal feed additive, such as in an amount of at least about 0.1% by weight of the feed, and in at least one embodiment, in an amount of about 0.1% to about 2% by weight of the feed. In an alternative embodiment, the feed additive is added to the water or pond, wherein the composition is administered in an amount effective to inactivate EHP spores present in shrimp culture water and shrimp pond soil sediment.

[0109] After describing the present invention by reference to specific compositions, theories of effectiveness, etc., it will be apparent to those skilled in the art that the present invention is not limited to this exemplary embodiment or mechanism and can be modified without departing from the scope or spirit of the present invention as defined by the appended claims. All such obvious modifications and variations are intended to be included within the scope of the present invention as defined by the appended claims. The claims are intended to cover the claimed components and steps as long as they effectively achieve their intended purpose in any order, unless the context clearly indicates the contrary.

[0110] It should also be understood that minor dosage and formulation modifications can be made to the compositions and ranges expressed herein and still remain within the scope and spirit of the present invention.

[0111] It should also be understood that the formulations and methods shown in the figures and described in the following specification are merely exemplary embodiments of the inventive concepts defined in the appended claims. Accordingly, the specific dimensions and other physical characteristics of the embodiments disclosed herein are not to be considered as limiting unless the claims expressly state otherwise. When a numerical range is provided, each intermediate value (to the tenth of the unit of the lower limit) between the upper and lower limits of the range, as well as any other stated or intermediate value within the range, is covered within the scope of this disclosure. The upper and lower limits of these smaller ranges can be independently incorporated into the smaller ranges and are also covered within the scope of this disclosure, subject to the specific limitations excluded from the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included within the scope of this disclosure. All ranges and parameters disclosed herein (including, but not limited to, percentages, parts, and ratios) should be understood to cover any and all sub-ranges subsumed therein and all numerical values between the endpoints. For example, the range of "1 to 10" should be considered to include any and all sub-ranges starting with a minimum value of 1 or greater and ending with a maximum value of 10 or less (e.g., 1 to 6.1 or 2.3 to 9.4), as well as each integer (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) included within the range.

[0112] In this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. All combinations of method steps or process steps used herein can be performed in any order unless otherwise specified or the context of the recited combination clearly implies the contrary order.

[0113] Insofar as the terms "comprising", "including", or "having" are used in the specification or claims, their meaning is similar to the meaning of the term "containing" as used as a transitional word in the claims and is intended to be inclusive. Additionally, insofar as the term "or" (e.g., A or B) is used, it is intended to mean "A" or "B" or both "A" and "B". When the applicant intends to mean "only A or B but not both", the term "only A or B but not both" or a similar construction will be used. Accordingly, the use of the term "or" herein is inclusive and not exclusive. Further, insofar as the terms "in" or "into" are used in the specification or claims, they are intended to also additionally mean "on" or "upon". In this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

[0114] The foregoing description has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Other alternative processes and methods that would be apparent to one of ordinary skill in the art are also contemplated as being included within the invention. This description is only an example of an embodiment. It is understood that any other modifications, substitutions, and / or additions can be made within the intended spirit and scope of the present disclosure. From the foregoing, it can be seen that the exemplary aspects of the present disclosure achieve at least all of the intended objectives.

Claims

1. A method for controlling the spread of EHP infection and related diseases in shrimp, the method comprising administering to the shrimp a composition containing an amount of thymol effective to reduce the EHP infection rate.

2. The method according to claim 1, wherein the thymol is present in an amount of at least 100 ppm.

3. The method according to claim 1, wherein the thymol is derived from Monarda didyma, Monarda fistulosa or Nigella sativa.

4. The method according to claim 1, the method further comprising one or more essential oils selected from Monarda oil, oregano oil, clove oil and cinnamaldehyde.

5. The method according to claim 1, wherein the composition comprises thymol in an amount of about 0.01% to about 100% by weight, carvacrol in an amount of about 0 to about 70% by weight, α-terpineol in an amount of about 0 to about 70% by weight, cinnamaldehyde in an amount of about 0 to about 30% by weight and eugenol in an amount of 0 to about 30% by weight.

6. The method according to claim 1, wherein the composition is added to the shrimp diet as an animal feed additive.

7. The method according to claim 1, wherein the composition is added to water or a pond, wherein the composition is administered in an amount effective to inactivate EHP spores present in shrimp culture water and shrimp pond soil sediment.

8. The method according to claim 1, wherein the composition is added to the feed in an amount of about 0.1% to about 2% by weight of the feed.

9. The method according to claim 1, wherein the composition is added to the feed in an amount of at least about 0.1% by weight of the feed.

10. A method for inactivating EHP spores, the method comprising administering to the shrimp a composition containing an amount of Nigella sativa seed extract / oil and one or more essential oil / extracts effective to inactivate EHP spores and reduce the EHP infection rate.

11. The method according to claim 10, wherein the one or more essential oils are selected from Monarda oil, oregano oil, clove oil and cinnamaldehyde / cinnamon oil.

12. The method according to claim 10, wherein the composition comprises thymol in an amount of about 0.01% to about 100% by weight, carvacrol in an amount of about 0 to about 70% by weight, α-terpineol in an amount of about 0 to about 70% by weight, cinnamaldehyde in an amount of about 0 to about 30% by weight and eugenol in an amount of 0 to about 30% by weight.

13. The method according to claim 10, wherein the thymol is derived from Monarda, Monarda fistulosa or Nigella sativa.

14. The method according to claim 10, wherein the composition is added to the shrimp diet as an animal feed additive.

15. The method according to claim 10, wherein the composition is added to water or a pond, wherein the composition is administered in an amount effective to inactivate EHP spores present in shrimp culture water and shrimp pond soil sediment.

16. The method according to claim 10, wherein the composition is added to the feed in an amount of about 0.1% to about 2% by weight of the feed.

17. The method according to claim 10, wherein the composition is added to the feed in an amount of at least about 0.1% by weight of the feed.

18. A feed additive composition, the composition comprising an amount of thymol effective to inactivate EHP spores and reduce the EHP infection rate.

19. The feed additive according to claim 18, wherein the thymol is derived from Monarda didyma, Monarda fistulosa or Nigella foeniculum.

20. The feed additive according to claim 18, wherein the additive contains Nigella foeniculum extract or oil.

21. The feed additive according to claim 18, further comprising one or more essential oils selected from Monarda didyma oil, Origanum vulgare oil, Syzygium aromaticum oil and cinnamaldehyde / cinnamon oil.

22. The feed additive according to claim 18, wherein the composition contains an amount of thymol of about 0.01% to about 100% by weight, an amount of carvacrol of about 0 to about 70% by weight, an amount of α-terpineol of about 0 to about 70% by weight, an amount of cinnamaldehyde of about 0 to about 30% by weight and an amount of eugenol of 0 to about 30% by weight.

23. The feed additive according to claim 18, wherein the composition is added to the shrimp diet as an animal feed additive.

24. The feed additive according to claim 18, wherein the composition is added to water or a pond, and the composition is applied in an amount effective to inactivate EHP spores present in shrimp culture water and shrimp pond soil sediment.

25. The feed additive according to claim 18, wherein the composition is added to the feed in an amount of at least about 0.1% by weight of the feed.

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