JP5863839B2 - Vaccine against Vibrio infection in shrimp and method for producing the same - Google Patents

Description

  The present invention relates to a vaccine against Vibrio infection in shrimp and a method for producing the same.

  Vibrio disease caused by vibrio infection, which usually occurs when the host animal becomes weak, is considered a secondary opportunistic infection, and the occurrence of vibrio disease is associated with an increase in Vibrio spp. In aquaculture pond water (Lightner et al., 1998; Lavilla-Pitogo, et al., 1998). Vibrio species reported in the shrimp farming system include V. alginoliticus, V.M. angulararum, V.M. campbelli, V.M. damsella, V.M. fischeri, V.M. harveyi, V.H. logei, V.M. mediterani, V.M. ordalii, V.M. orientalis, V.M. parahaemolyticus, V. et al. pelagicus, V. et al. splendidus, V.M. There are vulnificus. Therefore, prevention of disease has become a major concern in shrimp farming. Antibiotics and other treatments are widely used in general post-infection clinical practice. However, the application of these therapeutic agents causes an increase in resistance to antibiotics, reduces the effectiveness of antibiotic treatments, creates residual accumulation in the tissue and environmental hazards, and creates public health problems ( karunasagar et al., 1994; Cabello et al., 2006).

  Immunological prevention such as immunostimulation, plant and microorganism-derived raw material vaccines, probiotics, etc. has become a promising and viable method for the prevention of pathogenic infections (Gatesoopup et al., 1999; Smith et al. 2003; Teunissen et al., 1998; Pereira et al., 2009). While immunostimulants are known to induce innate immunity, increase resistance to pathogens and alleviate immune suppression, prophylactic administration with vaccines provides protection against certain pathogens. Vaccines can be prepared by inactivating pathogens with formalin, ethanol, sodium sulfate, ammonium sulfite, fatty acids, and heat (Hossain et al., 2009).

  To date, the shrimp immune system is not yet fully understood. To date, shrimp, like other invertebrates, are able to protect themselves against harmful microorganisms by circulating blood cells (agranule cells (HC), small granule cells (semi-GC), granule cells (GC)). It has been reported that it relies solely on the innate immune system that plays an important role in defense for (Martin et al., 1985; Loker et al., 2004; Zhang et al., 2006). It is known that hematopoietic tissue (HPT) is responsible for the production and supply of blood cells (van de Braak et al., 2002). The shrimp immune system consists of a turn recognition system, a phenol oxidase precursor (proPO) activation system, release of antimicrobial peptides and lysozyme, and phagocytosis, nodule formation, encapsulation (Jiravanichpaisal et al., 2006; Rowley et al., 2007). ProPO is activated by melanin synthesis through a serine proteinase cascade by endogenous trypsin-like serine proteinase and becomes phenol oxidase (PO) (Cerenus et al., 2008; Soederhaell et al., 1998). A respiratory burst (RB) occurs during phagocytosis, releasing superoxide anions and other reactive oxygen species (ROS) (Bogan et al., 2000). The superoxide anion is eliminated by superoxide dismutase (SOD) and prevents damage to the host itself through the antioxidant system (Fridovich et al., 1995).

  Some recent studies have suggested the existence of specific memory within the innate immune system or the possibility of certain forms of adaptive immunity in insects and shrimp (Rowley et al., 2007; Arala-Chaves et al., 2000; Kurtz et al., 2004; Kurtz et al., 2005; Roth et al., 2009). Based on the presence of adaptive immunity or specific immune memory, several commercial vaccines have been developed for teleosts and shrimps (Sommerset et al., 2005; Powell et al., 2011). Furthermore, in crustaceans, a Down syndrome cell adhesion molecule (belonging to the immunoglobulin superfamily), also called Dscam, which plays an important role in “alternative adaptive immunity” was observed (Chou et al., 2009; Chou et al. , 2011; Wattansurorot et al., 2011). Some reports show that vaccination with formalin-inactivated Vibrio sp. Was effective against Vibrio disease in juvenile shrimp farming (Teunissen et al., 1998; Pereira et al., 2009; Itami et al., 1989; Itami et al., 1991; Itami et al., 1992; Alabi et al., 1999). However, little or no is known about the mechanism of action of shrimp administered formalin-inactivated pathogens and the immune response of shrimp secondary infected with specific pathogens. There is still a need for improved shrimp vaccines against Vibrio infections by examining shrimp immune response patterns.

  The objective of this invention is providing the vaccine with respect to the vibrio infection in shrimp, and its preparation method.

  In this study, heat inactivation alginoliticus (HVa) or formalin inactivated after primary exposure to Alginoliticus (FVa), followed by raw V. pneumoniae. Experiments were conducted to understand the immune response pattern of white shrimp that had undergone secondary exposure of arginoliticus (LVa). After primary exposure, HVa-administered shrimp showed a faster increase in immune response on day 1, while FVa-administered shrimp showed a slower increase in immune response on day 5, and after secondary exposure, HVa-administered shrimp and FVa Both doses of shrimp show vaccine efficacy, of which FVa doses show higher resistance to LVa, faster recovery of immune parameters, and higher HPT proliferation and mitotic index It was. Thus, both HVa and FVa can be used as vaccines to protect shrimp from Vibrio infections, and surprisingly HVa not only serves as a “vaccine” but also promotes the induction of specific recognition and memory It can also be used as an “immunoactive substance” or “adjuvant”. It has been shown that a combined mixture of FVa and HVa unexpectedly induces an improved and prolonged immune response to Vibrio infection (clearance efficiency> 40% and lasts for more than 42 days after vaccine).

  Accordingly, in one aspect, the invention provides a vaccine composition against infection caused by Vibrio in shrimp. The vaccine composition comprises heat-inactivated cells of Vibrio (heat-inactivated Vibrio cells) and formalin-inactivated cells of Vibrio (formalin-inactivated Vibrio cells), of which the heat-inactivated Vibrio cells and The formalin-inactivated vibrio cells are present in an effective amount that induces immunity specific for Vibrio in shrimp.

  In another aspect, the present invention provides the use of a mixture of heat-inactivated and formalin-inactivated vibrio cells for the production of a vaccine composition that is administered to shrimp to induce immunity specific for Vibrio. To do.

  In yet another aspect, the present invention provides the use of heat inactivated vibrio cells as an adjuvant to improve immunity of vaccines made from formalin inactivated vibrio cells.

  In yet another aspect, the present invention provides a vaccine composition comprising the step of mixing heat-inactivated vibrio cells with formalin-inactivated vibrio cells to form a mixture in an effective amount for inducing immunity specific for Vibrio bacteria. A method for manufacturing an object is provided.

  The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will be apparent from the following detailed description of several embodiments and the appended claims.

  The foregoing general description of the invention and the detailed description set forth below will be better understood by reference to the accompanying drawings, in which: For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. However, it should be understood that the invention is not limited to the precise arrangements and instrumentalities shown.

Heat inactivated by primary exposure (PE) Shrimp, formalin-inactivated shrimp that received Alginoliticus (HVa) Shrimp that received Alginoliticus (FVa), and shrimp that received exposure to seawater (MS) for control experiments, and raw V. pneumoniae 7 days after administration of HVa and FVa. 7-35 day HVa-PE and 7-35 day FVa-PE shrimp, and 7-28 day HVa-PE shrimp and 7-28 day FVa-PE of shrimp that have received secondary exposure (SE) of arginolyticus (LVa) It is an experimental plan which shows the phagocytosis and clearance with respect to LVa and live Bacillus subtilis (LBs) in both shrimps. Control shrimp and granule cells (HC) at 0 days, 0.5 days, 1 day, 3 days, 5 days and 7 days after being given seawater (marine salin, MS), HVa, FVa, LVa ( A is a graph showing granule cells (GC) (including semi-GC) (B), whole blood count (THC) (C). Each bar represents the mean and standard error (SE) from 6 shrimps. Data at the same time with different letters (a, b, c) are significantly different between treatments (p <0.05). Data of the same process with different letters (x, y, z) has a significant difference (p <0.05) between different elapsed times (days). Phenol oxidase (PO) activity (A) after 0 days, 0.5 days, 1, 3, 5, and 7 days of control shrimp and shrimp administered with seawater (MS), HVa, FVa, LVa It is a graph which shows respiratory burst (RB) (B), superoxide dismutase (SOD) activity (C), and lysozyme activity (D). See Figure 2 for statistical information. It is a graph which shows the relative immune parameter (%) of the shrimp which administered HVa, FVa, and LVa 0.5 days, 1 day, 3 days, 5 days, and 7 days after. Data with different letters (a, b, c) in HVa-administered shrimp (mean ± SE), Data with different letters (r, s, t) in FVA-administered shrimp (mean ± SE), LVa administration Data (mean ± SE) with different letters (x, y, z) in shrimp are significantly different (p <0.05) between different elapsed times (days). (A) Light micrograph of H & E-stained hematopoietic tissue (HPT) of control shrimp (a), HVa-administered shrimp (b) and FVa-administered shrimp (c) 5 days later. A larger number of mitotic cells (arrows) was observed in shrimp administered with HVa and shrimp administered with FVa. (B) The percentage of proliferating cells (%) in HPT of FVa-administered shrimp and HVa-administered shrimp was significantly higher than that of control shrimp. (C) Fluorescence micrographs of propidium iodide-stained HPT of control shrimp (a), HVa-administered shrimp (b) and FVa-administered shrimp (c) 5 days later. A larger number of mitotic cells (arrows) was observed in shrimp administered with HVa and shrimp administered with FVa. (D) The mitotic index, expressed as the percentage of dividing cells in HPT of FVa-treated shrimp and HPT of HVa-treated shrimp, was significantly higher than that of control shrimp. Each bar represents the mean and standard error (SE) from 6 different images. Data with different letters are significantly different between treatments (p <0.05). Scale = 20 μm. It is a graph which shows the survival rate when attacking the control shrimp without viable bacteria attack, and the control shrimp and HVa and FVa administration shrimp 7 days later with LVa of the number of bacteria of 8.0 × 10 ⁶ cfu shrimp- ¹ . Each data point represents the mean and standard error (SE) of 3 replicates (10 replicates each). Data with different characters in the same period have a significant difference between treatments (p <0.05). The control shrimp and shrimp 7 days after HVa and FVa administration were challenged with LVa having a bacterial count of 3.8 × 10 ⁵ cfu shrimp- ¹ at 0, 0.5, 1, 3, 5, 7 It is a graph which shows the non-granule cell (HC) (A), granule cell (GC) (including semi-GC) (B), and the total blood count (THC) (C) after a day. See Figure 2 for statistical information. The control shrimp and shrimp 7 days after HVa and FVa administration were challenged with LVa having a bacterial count of 3.8 × 10 ⁵ cfu shrimp- ¹ at 0, 0.5, 1, 3, 5, 7 It is a graph which shows the phenol oxidase (PO) activity (A), respiratory burst (RB) (B), superoxide dismutase (SOD) activity (C), and lysozyme activity (D) after a day. See Figure 2 for statistical information. (A) It is a graph which shows the relative immunity parameter (%) after attacking LVa to the control shrimp and the shrimp 7 days after HVa and FVa administration. Data with different letters (x, y, z) in the control shrimp (mean ± SE), data with different letters (r, s, t) in the HVa-treated shrimp (mean ± SE), shrimp with FVa Data (mean ± SE) with different letters (a, b, c) in are significantly different between elapsed times (days) (p <0.05). (B) It is a graph which shows the relative value of HVa administration shrimp and FVa administration shrimp with respect to control shrimp. Data with different letters (r, s, t) for shrimp HVa administration and data with different letters (a, b, c) for shrimp FVa administration have a significant difference between elapsed times (days). Yes (p <0.05). (A) Light micrograph of H & E-stained hematopoietic tissue (HPT) 3 days after LVa challenge to control shrimp (a), HVa-administered shrimp (b) and FVa-administered shrimp (c) 7 days later. A larger number of mitotic cells (arrows) was observed in the FVa-treated shrimp. (B) The percentage of proliferating cells (%) in HPT of shrimp 3 days after LVa challenge on FVa-administered shrimp 7 days later was significantly high. (C) Fluorescence micrographs of propidium iodide-stained HPT three days after LVa challenge to control shrimp (a), HVa-administered shrimp (b) and FVa-administered shrimp (c) 7 days later. A larger number of mitotic cells (arrows) was observed in the FVa-treated shrimp. (D) The mitotic index expressed as the percentage of dividing cells in HPT 3 days after LVa challenge to FVa-administered shrimp 7 days later was significantly higher. Scale = 20 μm. See Figure 5B for statistical information. It is a photograph which shows the aggregation activity after performing LVa challenge to control shrimp (A) and HVa administration shrimp (B) and FVa (C) 7 days after. FVa and HVa administered shrimp showed higher aggregation activity. Aggregated “bacterial clusters” were phagocytosed by agranular cells (HC) (D) and small granule cells (SGC) (E), both 7 day HVa-PE shrimp and 7 day FVa-PE shrimp And phagocytosis by both HC (F) and SGC (G) is 14 days of HVa-PE shrimp and 14 days of FVa-PE shrimp, and phagocytosis by both HC (H) and SGC (I) is 21 Observed on day HVa-PE shrimp and 21 day FVa-PE shrimp. Different bacterial intacts, including LVa (J), HVa (K), FVa (L), were stained by the Liu staining method (Liu's staining). Scale = 5 μm (A to I), 2 μm (J to L). Control shrimp, phagocytic ability (A), phagocytosis coefficient (B), clearance ability (C) after LVa challenge to HVa-PE shrimp from 7 to 35 days and FVa-PE shrimp from 7 to 35 days It is a graph to show. Control shrimp, phagocytic ability (D), phagocytosis coefficient (E), clearance after 7-28 days HVa-PE shrimp and 7-28 days FVa-PE shrimp attacked with Bacillus subtilis (LBs) It is a graph which shows capability (F). Data for the same exposure time with different letters (a, b, c) are significantly different between different treatments (p <0.05). It is a graph which shows the clearance ability after performing LVa attack to the control shrimp and the (HVa + FVa) -PE shrimp 7 to 42 days later. Data for the same exposure time with different letters (a, b) are significantly different between different treatments (p <0.05).

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood in ordinary skill in the art to which this invention belongs.

  As used herein, “a” and “one” (articles “a” and “an”) refer to one or more (ie, at least one) grammatical objects thereof. . For example, “an element” means one element or more than one element.

  As used herein, the term “vaccine” or “vaccine composition” refers to an immunogenic composition of an antigenic substance (eg, used to elicit immunity specific for that antigenic substance in a recipient). Can be changed. Thus, after a subject has been vaccinated or immunized with a vaccine, the subject forms immunity that specifically recognizes the antigen of the vaccine and kills the pathogen when further exposed to the pathogen or Inactivate. In shrimps, immunization or vaccination can be performed by injection or immersion. Furthermore, immunity in shrimp can be determined based on various parameters such as the number of cells of blood cells, such as agranular cells (HC), small granule cells (semi-GC), granule cells (GC), phenol oxidation Enzyme (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, lysozyme activity, cell proliferation, hematopoietic tissue (HPT) mitotic index, phagocytic ability, clearance ability. Standard tests for measuring these parameters for shrimp are known within the skill of the artisan.

  As used herein, “inactivated” or “inactivated” refers to a vaccine made from a pathogenic bacterium, so that the infectivity, toxicity, or pathogenicity of the pathogenic bacterium in the vaccine is, for example, heat or formalin, It means that the destruction process is performed by a method such as ultraviolet light irradiation.

  According to the present invention, a mixture of heat-inactivated vibrio cells and formalin-inactivated vibrio cells can induce enhanced or prolonged immune responses to vibrio infection in shrimp compared to formalin-inactivated vibrio cells alone. It has been shown to act as an improved vaccine against infection.

  Accordingly, in one aspect, the invention provides a vaccine composition against infection caused by Vibrio in shrimp. The vaccine composition comprises heat-inactivated cells of Vibrio (heat-inactivated Vibrio cells) and formalin-inactivated cells of Vibrio (formalin-inactivated Vibrio cells), of which the heat-inactivated Vibrio cells and The formalin-inactivated vibrio cells are present in an amount effective to induce immunity specific for Vibrio in shrimp. The present invention also provides the use of a mixture of the heat-inactivated vibrio cells and the formalin-inactivated vibrio cells in the manufacture of a vaccine composition for administration to shrimp to induce immunity specific to the Vibrio bacteria.

  In some embodiments, the Vibrio bacteria include V. alginoliticus, V.M. angulararum, V.M. campbelli, V.M. damsella, V.M. fischeri, V.M. harveyi, V.H. logei, V.M. mediterani, V.M. ordalii, V.M. orientalis, V.M. parahaemolyticus, V. et al. pelagicus, V. et al. splendidus, V.M. vulnificus is included, but is not limited thereto. This bacterium can be commercially available or can be isolated from moribund shrimp based on standard procedures reported in the prior art. The bacteria can be cultured overnight in a medium at about 28 ° C. The medium used for recent cultures can be TB (Terrific broth) or TSB (tryptic soy broth), 2% NaCl, and amino acids such as phenylalanine, tyrosine, tryptophan, paraaminobenzoic acid, parahydroxybenzoic acid, etc. Is added.

  In some embodiments, the shrimp includes but is not limited to vaname shrimp, black tiger shrimp, tiger shrimp, red shrimp, indian shrimp, reed shrimp, red tail shrimp, tiger shrimp.

  Heat-inactivated vibrio cells and formalin-inactivated vibrio cells can be produced according to various procedures known to those skilled in the art. In some embodiments, the production of heat-inactivated vibrio cells involves culturing the bacteria, harvesting and resuspending them in sea water, and then suspending the bacterial suspension at a temperature above 80 ° C., preferably 90 °. More than 100 ° C., more preferably 100 ° C. or more, preferably 120 ° C. with a dish, for a sufficient time, for example, 5 minutes or more, preferably 10 minutes or more, more preferably 15 minutes or more, heating in a heating box, heating tank, or autoclave To do. In certain embodiments, the bacterial suspension is autoclaved at 121 ° C. for 20 minutes. In another embodiment, the production of formalin-inactivated vibrio cells may be performed at an effective concentration of the bacterial suspension for a sufficient time, eg, about 12 to about 96 hours, preferably about 24 to 48 hours, more preferably about 24 hours. Treated with formalin. In some examples, the procedure for bacterial treatment with formalin is performed at about 4 ° C to 40 ° C. In some embodiments, the effective concentration of formalin for treating the bacterial sample ranges from about 0.1% to about 2% (v / v), such as 0.1% (v / v). 0.2% (v / v), 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), 0.6% (v / v) , 0.7% (v / v), 0.8% (v / v), 0.9% (v / v), 1.0% (v / v), or up to 2.0% (v / v) v). In certain embodiments, the bacterial suspension is obtained and formalin is added to a final concentration of 1% formalin and incubated at 4 ° C. for 24 hours. In addition, the bacterial sample is treated with formalin and then centrifuged or filled to recover the bacterial pellet and washed with culture to remove residual formaldehyde.

In some embodiments, the vibrio cells in the vaccine composition of the invention (the heat-inactivated vibrio cells + the formalin-inactivated vibrio cells) are present in an amount of 10 ⁵ -10 ⁹ CFU / ml. In particular, the heat-inactivated vibrio cells + the formalin-inactivated vibrio cells in the vaccine composition of the present invention are present at a CFU ratio of 0.1: 1 to 1: 0.1 (HVa: FVa).

In some embodiments, methods of administration include, but are not limited to, injection and immersion. In one embodiment, for use in injection administration, shrimp were injected with the vaccine composition of the invention at a dose of 10 ⁴ -10 ⁸ CFU per tail. In another embodiment, for use in administration by immersion, a vaccine stock made by immersion with the vaccine composition of the present invention and administered the vaccine to shrimp, eg, made of sterile aged brackish water or seawater in a 1 L tank Can be achieved by diluting the solution in 20 L of water to achieve a concentration of 10 ² -10 ⁵ CFU / ml, soaking time is about 30 minutes or more, for example 1 hour, 2 hours, 3 hours, or more This can be done.

  According to the present invention, a mixture of heat-inactivated vibrio cells and formalin-inactivated vibrio cells, as demonstrated by the following experiment, compared to the case of heat-inactivated vibrio cells or formalin-inactivated vibrio cells alone, It can be used as an improved vaccine that induces a significant immune response to vibrio infection in shrimp. In particular, the clearance rate induced in the mixture of heat-inactivated and formalin-inactivated vibrio cells is as high as at least 40% and lasts from 28 days to 42 days. Immunity persistence is important in shrimp farming. This is because the shrimp farming phase is about 4 to 4.5 months, and the immunity lasts longer, so the number of vaccine administrations (re-strengthening) can be reduced and the cost of shrimp farming can be reduced. is there. This connection suggests that in vaccine compositions, heat-inactivated vibrio cells serve as adjuvants that promote the induction of immune responses in shrimp.

  Accordingly, the present invention also provides the use of heat inactivated vibrio cells as an adjuvant to improve immunity of vaccines made from formalin inactivated vibrio cells.

  As used herein, an “adjuvant” refers to a substance or agent that when administered with an antigen enhances the recipient's immune response to that antigen.

  In one embodiment, the heat-inactivated vibrio cells are mixed with the formalin-inactivated vibrio cells to induce an enhancement and prolongation of the immune response against Vibrio bacteria as compared to the formalin-inactivated vibrio cells alone. Form.

  In yet another aspect, the present invention provides a method of making a vaccine composition against vibrio infection in shrimp, comprising the step of mixing heat inactivated vibrio cells with formalin inactivated vibrio cells. Usually, seawater is used to prepare the vaccine of the present invention. Other excipients and carriers can also be used in preparing the vaccine. In particular, the method of the present invention can be performed by simply mixing heat-inactivated and formalin-inactivated vibrio cells to form a homogeneous mixture, and no additional adjuvant components are required.

In certain embodiments, a method of making a vaccine composition against Vibrio infection in shrimp of the invention comprises:
(A) culturing and growing Vibrio cells;
(B) collecting the proliferated cells and dividing the collected cells into a first part and a second part;
(C) Inactivating the first part of the collected cells by heating to obtain heat-inactivated vibrio cells, and inactivating the second part of the collected cells with formalin to obtain formalin-inactivated vibrio cells. When,
(D) mixing heat-inactivated vibrio cells with formalin-inactivated vibrio cells to obtain a vaccine composition against vibrio in shrimp;
including.

In some embodiments, the heat-inactivated vibrio cells and the formalin-inactivated vibrio cells are mixed to provide an amount of 10 ⁵ -10 ⁹ CFU / ml in the vaccine composition. In some embodiments, the heat-inactivated vibrio cells and the formalin-inactivated vibrio cells in the vaccine composition of the present invention are present in a CFU ratio of 0.1-1 to 1: 0.1 (HVa: FVa). To do.

  In one particular embodiment, the method of making a vaccine composition of the invention does not include the step of adding additional components as an adjuvant.

  Thus, one of the advantages of the method of the invention is the reduction in the number of steps carried out without the addition of further adjuvants, and some of the known adjuvants (eg aluminum or oil) within the skill of the person skilled in the art. Base adjuvants) can be expensive and require special safety concerns and quality control, which can lead to higher costs. In other words, the method of preparing a vaccine composition against Vibrio infection by mixing both heat-inactivated vibrio cells and formalin-inactivated vibrio cells of the present invention is simple and cost-effective, and can be applied to large-scale production. There is sex.

  The following examples further illustrate the present invention. These examples are for illustrative purposes and do not limit the invention. A person skilled in the art may make many changes to the specific embodiments disclosed based on the disclosure of the present invention and obtain similar or similar results without departing from the spirit and scope of the present invention. It will be possible.

Some term abbreviations are shown below.

  In this experiment, the immune parameters of shrimp that received LVa, HVa, and FVa were investigated, and the survival rate and immune parameters of shrimp after LVa challenge that received LVa 7 days after the administration of each HVa and FVa investigated. Blood cell proliferation and HPT (hematopoietic tissue) mitotic index of control shrimp, HVa shrimp and FVa shrimp were examined before and after LVa challenge. Blood profile, PO activity, RB, SOD activity, lysozyme activity were used as indicators of immune parameters (Rodriguez et al., 2000). The phagocytosis and clearance of shrimp LVa challenged 7-35 days after receiving HVa and FVa were investigated. In addition, the phagocytosis and clearance of shrimp attacked by Bacillus subtilis (LBs) were investigated 7-28 days after receiving HVa and FVa. Finally, the clearance of LVa challenged shrimp was investigated 7-35 days after receiving a combination of HVa and FVa.

  1. Materials and methods

1.1. White Shrimp Banana Shrimp White Shrimp Banama Shrimp was provided to the laboratory by the University Marine Animal Center adjacent to the coast of Keelung, Taiwan. Shrimp were placed in an indoor glass fiber circulator tank containing aerated seawater (35% salinity) and allowed to acclimate for 3 weeks until the experiment began. During the acclimatization period, shrimp were fed twice a day with a 3% shrimp formula feed (Tairou Feed Company, Tainan, Taiwan).

1.2. Production of LVa and LBs In this experiment, V. LVs isolated from dying vaname shrimp were isolated. The pathogenic strain of Alginoliticus was used (Liu et al., 2004). Bacteria were cultured in Tryptic Soy Broth (TSB with 2% NaCl, Difco, Sparks, MD, USA) for 24 hours at 28 ° C. and then centrifuged at 7155 × g for 20 minutes at 4 ° C. (Yeh et al., 2009). The supernatant is removed and the bacterial pellet is resuspended in seawater (MS) at 1.9 × 10 ⁷ and immunized as each bacterial suspension with 4.0 × 10 ⁸ colony forming units (cfu) ml ^−1. Prepared for parameter investigation and viable challenge test. Bacillus subtilis (BCRC10448) obtained from Bioresource Collection and Resource Center (Hsinchu City, Taiwan) was cultured in the same manner as described above, and a bacterial suspension was prepared at 4.0 × 10 ⁸ cfu ml ⁻¹ for a live bacterial challenge test. It was.

1.3. Preparation of HVa and FVa The bacterial suspension (1.9 × 10 ⁷ cfu ml ⁻¹ ) was centrifuged at 4500 × g for 10 minutes at 4 ° C., and the bacterial pellet was washed with MS and then centrifuged again. Separation and culture broth were removed. Finally, the pellet was resuspended in the same volume of MS and autoclaved at 121 ° C. for 20 minutes. The bacterial suspension after autoclaving was used for heat inactivation treatment. Another bacterial suspension (1.9 × 10 ⁷ cfu ml ⁻¹ ) was centrifuged and a pellet was obtained as described above. Formalin was added to the bacterial suspension in MS to a final concentration of 1% formalin and incubated at 4 ° C. for 24 hours. The bacterial suspension was centrifuged at 4500 × g for 10 minutes at 4 ° C. to recover the bacterial pellet, then washed with MS and centrifuged again to remove residual formaldehyde. The bacterial suspension after formalin treatment was used for formalin inactivation treatment.

1.4. Experimental design Seven studies were conducted. Experiments were designed based on traditional immunization to test primary exposure (PE) and secondary exposure (SE) to specific pathogens (Figure 1). (1) The immune parameters of control shrimp, MS-PE shrimp, LVa-PE shrimp, HVa-PE shrimp and FVa-PE shrimp were investigated over 0-7 days. (2) HPT blood cell proliferation and mitotic index of control shrimp, HVa-PE shrimp, and FVa-PE shrimp were measured on day 5. (3) Resistance of control shrimp, 7-day HVa-PE shrimp (shrimp 7 days after receiving primary exposure to HVa), 7-day FVa-PE shrimp (shrimp 7 days after receiving primary exposure to FVa) Were investigated after SE to LVa. (4) The immune parameters of control shrimp, 7-day HVa-PE shrimp, 7-day FVa-PE shrimp were investigated 0.5-7 days after SE to LVa. (5) HPT blood cell proliferation and mitotic index of control shrimp, 7-day HVa-PE shrimp, 7-day FVa-PE shrimp were examined 3 days after SE to LVa. (6) Aggregation, phagocytosis, and clearance of 7-35 day HVa-PE shrimp and 7-35 day FVa-PE shrimp that underwent SE to LVa were investigated. (7) The phagocytosis and clearance of 7-28 day HVa-PE shrimp and 7-28 day FVa-PE shrimp subjected to LBs challenge were investigated. The shrimp weight ranged from 10.21 to 12.63 g and the average was 11.42 ± 1.21 g (mean ± SD). There was no significant size difference between treatments. Only shrimp at the molting stage was used in the study. The molting stage was determined by examining the caudal limb where partial contraction of the epidermis could be identified (Chan SM et al., 1988). During the experiment period, the water temperature was 26 to 29 ° C., the pH was 7.89 to 8.27, the salinity was 33 ‰ to 35 ‰, and the dissolved oxygen amount (DO) was 5.68 to 6.84 mg L- ¹ .

1.5. Immune parameters of LVa-PE shrimp, HVa-PE shrimp, FVa-PE shrimp There are 5 treatments, each treatment at 5 different times (0, 0.5, 1, 3, 5, 7 days) Carried out in There were (1) control shrimp, (2) MS administration shrimp, (3) LVa, (4) HVa, and (5) FVa. Eight shrimps were used for each treatment. 20 μl of bacterial suspension (LVa, HVa, or FVa) was injected at a concentration of 1.9 × 10 ⁷ cfu ml ⁻¹ into the ventral sinus of each shrimp head and chest, resulting in 3.8 × 10 ⁵ It became cfu shrimp- ¹ . Shrimp without treatment served as the background control, and shrimp that received 20 μl of MS served as the positive control group. Hemolymph was collected after 0.5, 1, 3, 5, 7 days. Sampling of hemolymph and preparation of diluted hemolymph were performed according to the procedure described previously (Yeh et al., 2009). Briefly, hemolymph (300 μl) was collected individually from each shrimp abdominal sinus and diluted with 2700 μl of anticoagulant. Hemolymph anticoagulant mixture (diluted hemolymph) was placed in 4 tubes. The tubes were filled with 500, 1000, 1000, and 500 μl of diluted hemolymph, respectively, for measuring (1) blood cell count and RB, (2) PO activity, (3) SOD activity, and (4) lysozyme activity, respectively. Used.

1.6. Cell proliferation and mitotic index of HPT in control shrimp, HVa-PE shrimp, FVa-PE shrimp 5 days after PE There were three treatments, and 5 shrimp were used for each treatment. These shrimps were administered (1) MS (control), (2) HVa, (3) FVa. Shrimp were injected with MS, HVa, or LVa in the same manner as described above. Five days later, shrimp were sampled and each injected with vinblastine (V1377, Sigma) to inhibit mitosis. Briefly, shrimp were injected into the abdominal sinus with 60 μl (0.5 mg ml ⁻¹ ) vinblastine and released into normal seawater (van de Braak et al., 2002). After 12 hours, 3 ml of Davidson's fixative (30 ml of 95% ethanol, 20 ml of 37% formaldehyde, 10 ml of acetic acid, 30 ml of distilled water) was injected into the back of the shrimp head chest. Sampling, fixation, embedding of HPT, preparation of permanent slides and observation of HPT were performed according to the methods described previously (van de Braak et al., 2002; Siriustanaum et al., 2011; Bell et al., 1993). ).

The other set of HPT-containing slides was deparaffinized in xylene and rehydrated with several ethanol solutions and tap water. For RNA digestion, a drop of RNase A solution (1 mg L ⁻¹ ) was dropped on the slide, followed by staining with propidium iodide (PI) solution (0.2 mg ml ⁻¹ ) for 10 minutes. Washed for 45 minutes. Preparation of permanent slides, observation, and determination of HPT splitting index were performed according to the method described previously (Zhang et al., 2006; Sirirustanaum et al., 2011; Humason et al., 1979).

1.7. Resistance of control shrimp, 7-day HVa-PE shrimp, 7-day FVa-PE shrimp after SE to LVa
A total of four treatments were performed, one treatment without viable cell attack and 3 live cell attack treatments. (1) MS administration of PE, (2) 7 days after HVa administration, (3) LVa was administered as SE to shrimp 7 days after FVa administration, and these were classified into groups with viable cell attack. Each shrimp craniothoracic abdominal sinus was injected with 20 μl of HVa (1.9 × 10 ⁷ cfu ml ⁻¹ ) or FVa (1.9 × 10 ⁷ cfu ml ⁻¹ ) bacterial suspension, resulting in 3.8 × 10 ⁵ cfu shrimp- ¹ . As a control group with live bacteria challenge, shrimp were injected with the same amount of sterile MS. Seven days later, 20 μl of LVa was individually injected into the abdominal sinus of each shrimp head chest at 4.0 × 10 ⁸ cfu ml ⁻¹ , resulting in 8.0 × 10 ⁶ cfu shrimp- ¹ . Control shrimp without viable challenge were injected with the same amount of MS as PE and the same amount of MS as SE. Each group contained 30 shrimps. Experimental shrimp and control shrimp (10 tail tank- ¹ ) were kept in a 40L tank containing 20L (35 ‰) of seawater and replicated three times. Therefore, 120 fish [(3 × 3 × 10) + (1 × 3 × 10)] were used for this study. Survival of shrimp was examined once every 12 hours on the first day and once a day thereafter until the end of the experiment on the seventh day.

1.8. Control parameters after SE to LVa, 7-day HVa-PE shrimp, 7-day FVa-PE shrimp immune parameters (1) Shrimp administered MS (control shrimp), (2) HVa after 7 days Three treatments of shrimp (7-day HVa-PE shrimp) and (3) FVa-shrimp shrimp 7 days later (7-day FVa-PE shrimp) were performed, and each treatment was conducted for 5 different periods (0.5, 1 3, 5, 7). Eight shrimps were used for each treatment and period. In addition, 8 untreated shrimps were used to obtain background values. Experimental shrimp were individually injected with 20 μl of HVa or FVa (1.9 × 10 ⁷ cfu ml ⁻¹ ), resulting in release of 3.8 × 10 ⁵ cfu shrimp- ¹ into normal seawater. Control shrimp were injected with 20 μl of MS. Seven days later, shrimp were individually injected with 20 μl of LVa at 1.9 × 10 ⁷ cfu ml ⁻¹ resulting in 3.8 × 10 ⁵ cfu shrimp- ¹ . Hemolymph was collected individually at 0.5, 1, 3, 5, 7 days after LVa challenge. Sampling of hemolymph and preparation of diluted hemolymph were performed as described above.

1.9. Measurement of immune parameters A drop of diluted hemolymph from the first tube is placed in a hemacytometer and HC, GC (semi-) using a phase contrast inverted microscope (Leica DMIL, Leica Microsystems, Wetzlar, Germany) GC) and whole blood count (THC) were measured. The remaining diluted hemolymph mixture was used for later testing.

  As previously described (Soderhall et al., 1979), with some modifications, total PO activity is measured spectrophotometrically by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA). Measured in Briefly, 1000 μl of diluted hemolymph from the second tube was centrifuged at 800 × g for 20 minutes at 4 ° C. Details of the measurement have been described previously (Yeh et al., 2009).

  Blood cell RB was quantified using the reduction of nitroblue tetrazolium (NBT) versus formazan as a measure of superoxide anion, as previously described (Bell et al., 1993). Briefly, 100 μl of diluted hemolymph from the first tube was triplicated into a 96-well microplate previously coated with 100 μl poly L-lysine solution (0.2%) to enhance cell adhesion. Noted. Details of the measurement have been described previously (Yeh et al., 2009).

  SOD activity was measured by its ability to inhibit superoxide radical-dependent reactions using the Ransod kit (Randox, UK). Briefly, 1000 μl of diluted hemolymph from the third tube was centrifuged at 800 × g for 20 minutes at 4 ° C. Details of the measurement have been described previously (Yeh et al., 2009; Biagini et al., 1995).

  Lysozyme activity was determined according to the method as previously described (Ellis et al., 1990). Briefly, 500 μl of diluted hemolymph from the fourth tube was centrifuged and the precipitate was mixed with 1 ml (0.02%) Micrococcus lysodeikticus (Sigma, St. Louis, Mo., USA). Details of the measurement have been described previously (Ellis et al., 1990; Tayag et al., 2010).

1.10. Cell growth and mitotic index of control shrimp, 7-day HVa-PE shrimp, 7-day FVa-PE shrimp after 3 days SE to LVa Three treatments were performed with 5 shrimp in each treatment. Shrimp received (1) MS, (2) HVa, and (3) FVa as PE for 7 days. Eight shrimps were used for each treatment and time. Shrimp were individually injected with 20 μl HVa or FVa (1.9 × 10 ⁷ cfu ml ⁻¹ ), resulting in 3.8 × 10 ⁵ cfu shrimp- ¹ released into normal seawater (35% salinity) for 7 days It was done. Shrimp were administered LVa as SE, and after 3 days the shrimp were sampled and individually injected with vinblastine (V1377, Sigma) to inhibit mitosis. The procedures for subsequent vinblastine injection, HPT fixation, slide staining, creation of permanent slides, observation of cell proliferation and mitotic index were performed as described in Section 2.6.

1.11. Phagocytosis and clearance capacity in HVa-PE shrimp and FVa-PE shrimp that have undergone SE to LVa, and phagocytosis and clearance in LVa-shrimp HVa-PE shrimp and FVa-PE shrimp LVa There are 3 treatments for SE, and after receiving administration of (1) MS, (2) HVa, (3) FVa as PE for 7, 14, 21, 28, 35 days, SE as LVa (3.8 Shrimp that received 3 × 10 ⁵ cfu shrimp- ¹ ) for 3 hours were used in experiments to observe aggregated “bacterial clusters”, phagocytosis and clearance. After 50 μl hemolymph was sampled and gently mixed with 50 μl anticoagulant, 100 μl fixative (1% paraformaldehyde) was added and incubated for 30 minutes. Fixed blood cells were spread on glass slides using cytospin (Thermo, Chandon, UK) at 1000 rpm for 10 minutes. The slide glass was stained with Mr. Liu A solution for 30 seconds and with Mr. Liu B solution for 60 seconds, and then washed with running water for 30 seconds. V. of different processing alginoliticus cells were stained as described above. Phagocytosis and clearance ability experiments were performed according to the method described previously (Tayag CM et al., 2010). Phagocytosis was defined as the phagocytic rate (PR) and expressed as PR (%) = [(phagocytic blood cells) / (total blood cells)] × 100. The phagocytic coefficient (PI) was expressed as PI = [(bacteria phagocytosed by phagocytic blood cells) / (phagocytic blood cells)]. The number of bacterial colonies in the control shrimp (MS) was expressed as a control group, and colonies of HVa-administered shrimp and FVa-administered shrimp were taken as the test group. The clearance ability was expressed as “100 − [(cfu of test group) / (cfu of control group)] × 100”. Three treatments were performed for comparison of LBs. Shrimp that received LBs attack (3.8 × 10 ⁵ cfu shrimp- ¹ ) for 3 hours after 7, 14, 21, 28 days after administration of (1) MS, (2) HVa, (3) FVa to shrimp Used for phagocytosis and clearance observation experiments. The phagocytic ability, phagocytosis coefficient, and clearance ability were carried out in the same manner as described above.

1.12. Preparation of a mixture of HVa and FVa A bacterial suspension (1.9 × 10 ⁷ cfu ml ⁻¹ ) was centrifuged at 4500 × g for 10 minutes at 4 ° C., the bacterial pellet washed with MS and centrifuged again And the culture medium was removed. Finally, the pellet was resuspended with the same amount of MS and autoclaved at 121 ° C. for 20 minutes. The bacterial suspension after autoclaving was used for heat inactivation treatment. Another bacterial suspension (1.9 × 10 ⁷ cfu ml ⁻¹ ) was centrifuged and a pellet was obtained as described above. Formalin was added to the bacterial suspension in MS to a final concentration of 1% formalin and incubated at 4 ° C. for 24 hours. The bacterial suspension was centrifuged at 4500 × g for 10 minutes at 4 ° C. to recover the bacterial pellet, then washed with MS and centrifuged again to remove residual formaldehyde. Formalin-treated bacterial suspension was used for formalin inactivation treatment. Mixtures of FVa and HVa is ^{FVa (1.9 × 10 7 cfu ml} -1) 1 aliquots to ^{HVa (1.9 × 10 7 cfu ml} -1) was added to one portion 2 content of FVa + HVa mixture (1 9.9 × 10 ⁷ cfu ml ⁻¹ ).

1.13. Statistical analysis One-way analysis of variance (ANOVA) was performed on all data. Where significant differences were shown at the 0.05 level, significant differences between treatments were investigated with SAS computer software (SAS Institute, Cary, NC, USA) using a multiple comparison (Tukey) test. Ratio data (resistance test) was normalized using an inverse sine transform prior to analysis. Statistical significance required p to be <0.05.

  2. Results

2.1. Immune parameters of LVa-PE shrimp, HVa-PE shrimp, FVa-PE shrimp Significant in HC, GC, THC, PO activity, RB, SOD activity, lysozyme activity in control shrimp and MS shrimp in different periods There was no difference. The HCa, GC, THC of shrimp administered LVa decreased after 0.5 days and remained below background values for 0.5-7 days after reaching the lowest level after 1 day. HCa shrimp HC, GC, and THC increased for 1 to 5 days, and then returned to the background value after 7 days. HC, GC, and THC of shrimp administered with FVa decreased after 0.5 days, but then increased and increased to the highest level on day 5 (FIG. 2).

  The LVa-administered shrimp had decreased PO activity, RB, SOD activity, and lysozyme activity, and were lower than the background values on the 5th to 7th days. The PO activity, RB, SOD activity, and lysozyme activity of shrimp administered with HVa increased significantly after 1 day, decreased slightly thereafter, and became similar to the background value after 7 days. The PO activity, RB, and SOD activity of shrimp administered with FVa decreased on the 0.5th day and then increased to reach the highest level on the 5th day, but returned to the background value by the 7th day. The lysozyme activity of FVa-administered shrimp remained unchanged on days 0.5-7 (FIG. 3).

  In order to clarify the innate immune response pattern of shrimp that received HVa, FVa, and LVa as PE, immune parameters are normalized to background values and displayed as relative ratio values (FIG. 4). The immune parameter pattern of shrimp administered with LVa decreased for 0.5-7 days. The immune parameter pattern of shrimp administered with HVa increased more rapidly on day 1 and then gradually decreased and returned to background values by day 7. The immune parameter pattern of shrimp administered with FVa decreased on 0.5 day, then increased and reached the highest value on 5th day. Shrimp receiving HVa, FVa, LVa showed different innate immune response patterns.

2.2. Cell proliferation and division index of HPT in HVa-PE shrimp and FVa-PE shrimp on day 5 after PE. H & E-stained HPT optical photographs of control shrimp, HVa-treated shrimp, and FVa-treated shrimp on day 5 were obtained. (Data not shown). The proportion of proliferating cells of HVa-administered shrimp and FVa-administered shrimp increased significantly (FIG. 5A). Fluorescence photographs of PI-stained HPT of control shrimp, HVa-shrimp, and FVa-shrimp shrimp on day 5 were obtained, and it was observed that the number of mitotic cells was large in HVa-shrimp and FVa-shrimp (data not shown) ). The mitotic index, expressed as the dividing cell ratio in HPT, was significantly higher in HVa and FVa-treated shrimp than that of control shrimp (FIG. 5B).

2.3. Control shrimp after LVa as SE, 7-day HVa-PE shrimp, 7-day FVa-PE shrimp All control shrimp without viable cell challenge survived. In contrast, mortality began to occur after 0.5 days in the viable challenge group. On day 3-7, there was no significant difference in survival rate between HVa-administered shrimp and FVa-administered shrimp. However, on days 5-7, the survival rate was higher for the shrimp administered with FVa (83.3%) than for shrimp administered with HVa (70%). The survival rate of the control shrimp was much lower at 40% on the 4th to 7th day after the viable bacterial challenge (FIG. 6).

2.4. Immune parameters of control shrimp, LVa-PE shrimp, 7-day HVa-PE shrimp, 7-day FVa-PE shrimp after LVa as SE Immune parameters of control shrimp decreased on days 5-7 after LVa as SE . The immune parameters of 7-day HVa-PE shrimp gradually increased on days 3-7 after LVa as SE, but remained below the background value. The HC, GC and THC of the 7th day FVa-PE shrimp were lower than the first to fifth day after LVa as SE, but increased after that, and similar to the background value on the third to seventh day after LVa as SE became. 7 day FVa-PE shrimp PO, RB, SOD, lysozyme activity increased on day 1 after LVa as SE, and values were similar to background values on days 0.5-7 after LVa as SE (FIGS. 7 and 8).

  In order to clarify the secondary immune response pattern of shrimp who received MS, HVa, FVa as PE followed by LVa as SE, immune parameters are normalized to background values and displayed as relative ratio values (FIG. 9A). The immune parameter pattern of control shrimp and 7-day HVa-PE shrimp decreased gradually to the first day after LVa as SE and then gradually increased, but the values were background values 1-7 days after LVa as SE Remained much lower. However, the immune parameter pattern of the 7-day FVa-PE shrimp decreased on the 0.5th day, but gradually increased on the 1st day, and the value was similar to the background value on the 3rd to 7th day after the LVa as SE. became. Further calculations were performed to normalize the relative values of 7-day FVa-PE shrimp and 7-day HVa-PE shrimp relative to control shrimp (FIG. 9B). Both 7-day and 7-day FVa shrimp immunity was elicited when faced with LVa as SE, but 7-day FVa shrimp showed a more efficient immune response than 7-day HVa shrimp.

2.5. Cell growth and mitotic index of HPT in LVa as SE, 3 days post-SE control shrimp, 7-day HVa-PE shrimp, 7-day FVa-PE shrimp 3 days after SE after LVa as SE Optical photographs of H & E-stained HPTs of the eye control shrimp, 7-day HVa-PE shrimp, 7-day FVa-PE shrimp are shown in FIG. 10A. After LVa as SE, the percentage of proliferating cells of 7-day FVa-PE shrimp was significantly higher than that of 7-day HVa-PE shrimp and control shrimp (FIG. 10B). FIG. 10C shows fluorescence photographs of PI-stained HPT of control shrimp, 7-day HVa-PE shrimp, and 7-day FVa-PE shrimp after LV as SE. It was observed that the number of mitotic cells (arrows) was large in FVa-administered shrimp. The split index of 7 day FVa-PE shrimp 3 days after SE was significantly higher than control shrimp and 7 day HVa-PE shrimp (FIG. 10D).

2.6. Phagocytosis and clearance capacity in HVa-PE shrimp and FVa-PE shrimp treated with LVa as SE, and phagocytosis and clearance in HVa-PE shrimp and FVa-PE shrimp attacked with LBs Control shrimp 11A to 11C show LVa administration 3 hours after blood cell staining of 7-day HVa-PE shrimp and 7-day FVa-PE shrimp. In HVa and FVa treatment, bacteria aggregated to form bacterial clusters and then attached to blood cells. In both treatments, phagocytosis of bacterial clusters by HC and SGC was observed (FIGS. 11D and 11E). Similar bacterial aggregation and phagocytic HC and SGC phenomena were observed for both 14-day HVa-PE shrimp and 14-day FVa-PE shrimp (FIG. 11F, FIG. 11G), and 21-day HVa-PE shrimp and 21-day FVa-PE shrimp. In both (FIGS. 11H, 11I). Intact of LVa, HVa and FVa was confirmed by the Liu staining method (Liu's staining).

  The phagocytic activity of 7-35 day FVa-PE shrimp and 7-21 day HVa shrimp receiving LVa as SE was significantly higher than that of control shrimp receiving LVa (FIG. 12A). The phagocytic coefficient of 7-35 day FVa-PE shrimp and 7-35 day HVa-PE shrimp that received LVa was slightly higher than that of control shrimp that received LVa (FIG. 12B). The clearance capacity of both treatments remained higher up to 28 days (FIG. 12C). No significant difference in phagocytic ability was observed between 7-28 day HVa shrimp, 7-28 day FVa shrimp, and control shrimp receiving LBs (FIG. 12D), but 7-28 days receiving LBs. The phagocytic coefficient of 28-day FVa-PE shrimp and 7-28 day HVa-PE shrimp was slightly higher than that of control shrimp that received LBs (FIG. 12E). The clearance capacity of 7-day HVa-PE shrimp and 7-day FVa-PE shrimp receiving LBs was significantly higher than that of control shrimp receiving LBs. However, the tendency for clearance ability declined after 14 days (FIG. 11F).

2.7. Clearance ability in HVa + FVa-PE shrimp Clearance ability in hemolymph of shrimp administered with a combination mixture of HVa and FVa (CFU ratio 1: 1) is shown in FIG. Shrimp were divided into 7 groups and injected with a combination mixture of HVa and FVa (CFU ratio 1: 1). Each group was then exposed to LVa at 7, 14, 21, 28, 35, and 42 days after receiving the combination mixture (HVa + FVa), respectively. Hemolymph was collected from shrimps for analysis of clearance capacity 3 hours after exposure to LVa. A low amount of LVa in the hemolymph suggests a high clearance capacity. As shown in FIG. 13, the combined mixture of HVa and FVa showed 40% clearance capacity from treatment to day 42. The immunity of shrimp induced by the combined mixture of HVa and FVa is improved compared to the fact that the results of HVa and FVa administered shrimp do not maintain higher ability after 28 days as shown in FIG. 12C , Extended (protection period is 42 days versus 28 days, 1.5 times longer).

3. Conclusion and discussion From the current research results, both HVa and FVa are It can be used as a vaccine to protect shrimp from SE to arginolyticus. FVa is a better candidate based on primary immunity pattern, secondary immunity pattern, enhanced phagocytosis, and strength of immunity induction, whereas HVa is an early increase in primary immunity, slight improvement and enhancement in secondary immunity pattern Based on the phagocytosis effected and some strength of immunity induction, it showed duality as both an immunostimulant and a vaccine. Thus, HVa can be used as an “immunoactivator” or “adjuvant” that promotes specific recognition and induction of memory. The combined mixture of FVa and HVa effectively acts as a “vaccine component” to modulate the immune response and enhance immunity against Vibrio disease in the shrimp farming industry.

  In summary, the immune parameters of HVa-administered shrimp increased early after 1 day, while the immune parameters of FVa-administered shrimp increased after 5 days, with both treatments showing higher HPT proliferation. FVa-treated shrimp showed higher resistance to LVa, faster recovery of immune parameters, and higher HPT proliferation and mitotic index 3 days after challenge. In addition, shrimp that received a combination mixture of HVa and FVa showed improved and prolonged immunity over shrimp administered HVa or FVa alone. Such a combined mixture of HVa and FVa is useful for the development of improved vaccines against Vibrio infections in the shrimp farming industry.

None

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Claims (11)

A shrimp vaccine composition against infection caused by Vibrio in shrimp, heat inactivated cells of the Vibrio (heat inactivated Vibrio cells) and the vibrio of formalin-inactivated cells (formalin-inactivated Vibrio cells) A vaccine composition for shrimp , wherein the heat-inactivated vibrio cells and the formalin-inactivated vibrio cells are present in an amount effective for inducing specific immunity against vibrio in shrimp object. The Vibrio bacterium is V. alginoliticus, V.M. angulararum, V.M. campbelli, V.M. damsella, V.M. fischeri, V.M. harveyi, V.H. logei, V.M. mediterani, V.M. ordalii, V.M. orientalis, V.M. parahaemolyticus, V. et al. pelagicus, V. et al. splendidus, V.M. Shrimp vaccine composition according to claim 1, characterized in that it is selected from the group comprising vulnificus. The shrimp vaccine composition according to claim 1 or 2, wherein the vaccine composition is for injection or immersion administration. 4. The heat-inactivated vibrio cell and the formalin-inactivated vibrio cell are present in the vaccine composition in a total amount of 10 < ⁵ > -10 < ⁹ > CFU / ml. Shrimp vaccine composition. 5. The heat-inactivated vibrio cell and the formalin-inactivated vibrio cell are present in the vaccine composition at a CFU ratio of 0.1: 1 to 1: 0.1, respectively. A shrimp vaccine composition according to claim 1. The shrimp, vannamei shrimp (Litopenaeus vannamei), black tiger shrimp (Penaeus monodon), prawns (Marsupenaeus japonic u s), Kouraiebi (Fenneropenaeus chinensis), Indoebi (F. indicus), Metapenaeus Ensis (Metapenaeus ensis), red tail shrimp (F Penicillatus), Shrimp vaccine (Macrobrachium rosenbergii), selected from the group comprising shrimp vaccine composition according to any one of claims 1-5. Heat-inactivated cells of Vibrio as an adjuvant for improving the immunity of a shrimp vaccine against infection caused by Vibrio in Vibrio, prepared from Vibrio formalin-inactivated cells (formalin-inactivated Vibrio cells) Use of (heat inactivated vibrio cells). The heat-inactivated vibrio cells are mixed with the formalin-inactivated vibrio cells to form a mixture that can induce a more enhanced and prolonged immune response against the vibrio bacteria compared to the formalin-inactivated vibrio cells alone. Use according to claim 7, characterized in that. The method for producing a shrimp vaccine composition as defined in any one of claims 1 to 6, comprising a step of mixing the heat-inactivated vibrio cells and the formalin-inactivated vibrio cells to form a mixture. . The method according to claim 9, wherein the method does not include adding an additional component as an adjuvant to the mixture.   For the production of a shrimp vaccine composition against infections caused by Vibrio in shrimp, effective for inducing specific immunity including enhanced and prolonged immune response against Vibrio in shrimp,
  Use of a combination of Vibrio heat-inactivated cells (heat-inactivated Vibrio cells) and Vibrio formalin-inactivated cells (formalin-inactivated Vibrio cells).

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