Abstract:
The present invention is concerned with the development of a vaccine against  Aeromonas hydrophila  for use especially in fish. The invention provides an immunogenic S-layer protein of approximately 50 kDa of  A. hydrophila  for use in the development of a vaccine, as well as the nucleic acid encoding said protein and vaccines comprising said protein or nucleic acid encoding said protein.

Description:
FIELD OF THE INVENTION  
       [0001]    The present invention is concerned with the development of a vaccine against  Aeromonas hydrophila  for use especially in fish. The invention provides an immunogenic S-layer protein of approximately 50 kDa of  A. hydrophila  for use in the development of a vaccine, as well as the nucleic acid encoding said protein and vaccines comprising said protein or nucleic acid encoding said protein. 
       BACKGROUND OF THE INVENTION 
       [0002]      Aeromonas hydrophila  is an important pathogen that has caused major loss in the aquaculture industry (Shotts et al., 1972; Olivier et al., 1981; Esteve et al., 1995) and attempts have been made to develop an effective vaccine (Lamers et al., 1985; Baba et al., 1988b; Leung et al., 1997; Rahman and Kawai, 2000). The effects of a number of inactivated whole cell (WC) vaccines have been reported and an increase in serum antibody levels against  A. hydrophila  was observed in common carp immersed in a preparation of heat inactivated  A. hydrophila  (Lamers et al., 1985). Kusuda et al. (1987) also found an increase in the concentration of total serum proteins, when common carp were immunised with formalin killed  A. hydrophila.  Rainbow trout immunised by injection, immersion and oral administration of killed  A. hydrophila,  have been shown to produce antibodies in their serum, bile, skin and gut mucus, and skin and muscle extracts (Loghothetis and Austin, 1994). A polyvalent vaccine containing heat killed WC and formalin inactivated extra cellular products (ECP) of  A. hydrophila  has also been tested in two Indian major carp species (rohu and mrigal), but it failed to protect the fish against bacterial challenge (Chandran et al., 2002a). 
         [0003]    Bacteria biofilms grown on the surfaces of nutrient flakes (chitin) have been used as oral administered heat inactivated biofilm vaccines against  A. hydrophila,  and have been reported to elicit a protective response in Indian major carp (catla and rohu) and common carp (Azad et al., 1999). The biofilm vaccines have been found to be retained for longer than free cell vaccines in the tissues of gut, spleen and kidney in Indian major carp (Azad et al., 2000a). Post  A. hydrophila  challenge, Catfish, fed with biofilm vaccines showed significantly higher serum agglutinating antibody titres and relative percentage survival (RPS) values compared to those fed with free cells (Nayak et al. 2004b). When grown as a biofilm, it was noted that there was a change in  A. hydrophila  antigenicity (Asha et al., 2004). Specifically, the authors found that the S-layer proteins were lost and the Lipopolysaccharide (LPS) of the bacteria contained an additional high molecular weight band. Asha et al. (2004) suggested that this high molecular weight LPS band might elicit a protective immune response when the biofilm was administrated as an oral vaccine. 
         [0004]    Considerable interest has been shown in bacterial OMP vaccines. Rahman and Kawai (2000) found that the OMPs of  A. hydrophila  elicited protection against an  A. hydrophila  challenge, and suggested that a vaccine based on selected OMP antigens may be effective. Munn (1994) suggested outer membrane components such as LPS could represent protective vaccine candidates for Gram-negative bacteria, while Dooley et al. (1986) reported that LPS of  A. hydrophila  posses highly immunogenic O polysaccharide chains of homogeneous length, and these were conserved both morphologically and antigenically in virulent isolates. The role of LPS in protection was also shown, in common carp vaccinated with crude LPS (Baba et al., 1988b). This protection appears to be based on cellular immunity rather than humoral immunity (Baba et al., 1988a). Similarly, Loghothetis and Austin (1996b) reported that LPS could be a major antigenic component of  A. hydrophila.    
         [0005]    Live WC cell vaccines have also been found to increase antibody responses in fish (Loghothetis and Austin, 1994). Other live vaccines, such as live attenuated (mutant) vaccines have also been explored for  A. hydrophila.  For example, growth-deficient mutants of  A. hydrophila  have been found to be promising live vaccine candidates in fish (Leung et al., 1997). An AroA mutant  A. hydrophila  strain was also investigated and found to be protective in rainbow trout (Moral et al., 1998). The vaccine was also found to elicit significant protection against  A. salmonicida  (Vivas et al., 2004b). Vivas et al. (2004c) suggested that this live aroA attenuated vaccine had a high level of safety compared with normal strains as it has a lower potential to survive in water. Other live vaccines have also been investigated, for example Catfish,  Clarias batrachus  immunised with plasmid free  A. hydrophila  mutants, showed an increased survival rate following challenge with wild bacteria compared to the control group (Majumdar et al., 2006). Mutant strains of  A. hydrophila  with a highly attenuated exoenzyme were also shown to confer protection in swordtail fish,  Xiphophorus helleri  (Liu and Bi, 2006). 
         [0006]    Although all the vaccines reported have shown varying degrees of increased immunity and protection, no commercial vaccine is available for  A. hydrophila  (Loghothetis and Austin, 1996b; Rahman and Kawai, 2000; Fang et al., 2004; Vivas et al., 2005). Over 90 established or provisional serogroups within the genus  Aeromonas  have been described, and the heterologous nature of  A. hydrophila,  both biochemically and serologically are still the greatest concern for developing an effective vaccine against  A. hydrophila  (Sakazaki and Shimada 1984; Stevenson, 1988; Khashe et al., 1996; Newman, 1993; Janda et al., 1994b; Leung et al., 1995). 
         [0007]    It is amongst the objects of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages. 
         [0008]    It is a further object to provide a vaccine against  A. hydrophila,  which desirably exhibits broad protection to many serogroups of  A. hydrophila.    
       SUMMARY OF THE INVENTION 
       [0009]    The present invention is based on the isolation of a protein of approximately 50 kDa (as determined by SDS-PAGE) from the S-layer of  A. hydrophila  and its use to raise an immune response in an organism. 
         [0010]    Thus, in a first aspect, the present invention provides a protein of approximately 50 kDa from the S-layer of  A. hydrophila  for use in raising an immune response in an organism. The protein may find particular application as a vaccine, but could also be used to obtain an immune serum for use in another organism. 
         [0011]    The 50 kDa protein is encoded by a nucleotide sequence derived from  A. hydrophila  (such as a T4 isolate), and has a molecular weight of about 50 kDa. In a particularly preferred embodiment of this aspect of the invention, the 50 kDa protein is encoded by the nucleotide sequence as shown in  FIG. 8  or  9 , representing the coding region of the immunogenic S-layer protein of  A. hydrophila  of the present invention, and having the amino acid sequence as shown. 
         [0012]    The present invention also provides an antigenic analog, fragment or modification of the polypeptide of  FIG. 8  or  9 . An antigenic analog, fragment, or modification of said polypeptide is one that generates an immune response in an organism, especially fish, against  A. hydrophila.  Antigenicity of a polypeptide can be evaluated in vitro by, for example, performing a Western blot on the purified polypeptide (for example, an affinity purified polypeptide) using polyclonal antisera from an animal, such as a rabbit that was vaccinated with at least an antigenic portion of the native  A. hydrophila  S-layer 50 kDa protein of the present invention. 
         [0013]    Antigenic analogs of the polypeptide according to  FIG. 8  or  9  include polypeptides having amino acid substitutions that do not eliminate polypeptide antigenicity in an organism, especially fish. Substitutes for an amino acid may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, praline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Examples of preferred conservative substitutions include Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free —OH is maintained; and Gln for Asn to maintain a free NH 2 . 
         [0014]    Fragments of said 50 kDa polypeptide of the invention include polypeptides containing deletions or additions of one or more contiguous or non-contiguous amino acids that do not eliminate the antigenicity of the protein fragment in an organism, especially fish are also contemplated. Fragments of said 50 kDa polypeptide contain at least about six amino acids, preferably at least about 10 amino acids, more preferably at least about 25 amino acids, up to about 50-75 amino acids. 
         [0015]    A modified 50 kDa protein of the present invention includes 50 kDa proteins that are chemically and enzymatically derivatised at one or more constituent amino acids, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, phosphorylation and the attachment of carbohydrate or lipid moieties, cofactors, and the like. 
         [0016]    The polypeptides of the present invention can be expressed from an appropriate nucleic acid molecule. The present invention therefore extends to a nucleic acid molecule capable of expressing the polypeptide, antigenic analog, fragment or modification thereof. The nucleic acid may comprise the sequence as shown in  FIG. 8  or  9  or an appropriate modification or fragment thereof. 
         [0017]    The nucleic acid molecule of the invention can be DNA, RNA, or a combination thereof, and can include any combination of naturally occurring, chemically modified or enzymatically modified nucleotides. The nucleic acid molecule can be equivalent to a polynucleotide fragment encoding the S-layer protein from  A. hydrophila  defined hereinabove, or it can include said polynucleotide fragment in addition to one or more additional nucleotides or polynucleotides. For example, the nucleic acid molecule of the invention can be a vector, such as an expression or cloning vector. 
         [0018]    A vector useful in the present invention can be circular or linear, single-stranded or double stranded, and can include DNA, RNA, or any modification or combination thereof. The vector can be a plasmid, a cosmid, or a viral vector, such as a bacteriophage. Preferably, the nucleic acid molecule of the invention takes the form of an expression vector that is capable of expression in an organism or in a cell of the organism, in culture or in vivo. An organism or cell in which the coding sequence of the vector can be expressed can be eukaryotic or prokaryotic, and can be, without limitation, a bacterium, a yeast, an insect, a protozoan, or animals, such as a fish or a mammal. Preferably, the vector is expressible in a fish and/or in a conventional protein expression system, including bacteria, such as  E. coli,  yeast, such as  Pischia pastoris,  mammalian cell culture or insect cells. 
         [0019]    When the vector is intended for use in bacterial, yeast, mammalian or insect expression systems, the coding sequences of the vector may be engineered to utilise the conventional genetic code rather than the  A. hydrophila  genetic code that is employed in the native  A. hydrophila  coding sequences. 
         [0020]    It should be understood that the nucleic acid molecule of the invention can be single-stranded or double-stranded, and further that a single-stranded nucleic acid molecule of the invention includes a polynucleotide fragment having a nucleotide sequence that is complementary to a nucleotide sequence that encodes said 50 kDa protein or portion thereof according to the invention. As used herein, the term “complementary” refers to the ability of two single stranded polynucleotide fragments to base pair with each other. 
         [0021]    Further, a single-stranded nucleic acid molecule of the invention also includes a polynucleotide fragment having a nucleotide sequence that is substantially complementary to a nucleotide sequence that encodes said 50 kDa protein or portion thereof according to the invention, or to the complement of the nucleotide sequence that encodes said 50 kDa protein or portion thereof. Substantially complementary polynucleotide fragments can include at least one base pair mismatch, such that at least one nucleotide present on a first polynucleotide fragment will not base pair to at least one nucleotide present on a second polynucleotide fragment, however the two polynucleotide fragments will still have the capacity to hybridize. The present invention therefore encompasses polynucleotide fragments which are substantially complementary. Two polynucleotide fragments are substantially complementary if they hybridize under hybridization conditions exemplified by 2×SSC (SSC: 150 mM NaCl, 15 mM trisodium citrate, pH 7.6) at 55° C. Substantially complementary polynucleotide fragments for purposes of the present invention preferably share at least about 85% nucleotide identity, preferably at least about 90%, 95% or 99% nucleotide identity. Locations and levels of nucleotide sequence identity between two nucleotide sequences can be readily determined using, for example, CLUSTALW multiple sequence alignment software. 
         [0022]    The invention further includes a nucleic acid molecule comprising a polynucleotide fragment that hybridises to at least a portion of the complement of the sequences provided by  FIG. 8  or  9  under standard hybridisation conditions, provided that the polynucleotide fragment encodes a polypeptide comprising at least an antigenic portion of the 50 kDa protein of the present invention. Standard hybridisation conditions are exemplified by 2×SSC (SSC: 150 mM NaCl, 15 mM trisodium citrate, pH 7.6) at 55° C. 
         [0023]    The present invention further provides a vaccine for use in preventing or controlling disease in fish caused by  A. hydrophila.  The vaccine may be a polypeptide or polynucleotide vaccine. Said polynucleotide vaccine comprises a polynucleotide fragment, preferably a DNA fragment, having a nucleotide sequence encoding an antigenic polypeptide comprising at least an antigenic portion of the S-layer protein of approximately 50 kDa from  A. hydrophila  as shown in  FIG. 8  or  9 . 
         [0024]    The polypeptide vaccine of the invention comprises the 50 kDa protein having amino acid sequence shown in  FIG. 8  or  9 , an antigenic analog, fragment, or modification of said protein. This type of vaccine is referred to herein as a “protein subunit vaccine” even if it contains the entire 50 kDa protein sequence. The 50 kDa protein or antigenic analog, fragment, or modification thereof for use in the protein subunit vaccine of the invention can be naturally occurring (i.e. isolated from  A. hydrophila ) or recombinant. A protein subunit vaccine of the invention is conveniently administered to fish using bath immersion, ingestion, topical administration, or direct injection, preferably intraperitoneal or intramuscular injection. A protein subunit vaccine formulated for oral administration can contain the polypeptide encapsulated in for example, a biodegradable polymer as described hereinafter. In addition, the protein subunit vaccine can be administered to an animal via a live vector, such as recombinant  Tetrahymena.    
         [0025]    The invention further provides a method for immunising freshwater fish or shellfish against  A. hydrophila  by administering to the fish/shellfish a protein subunit vaccine of the invention. 
         [0026]    The amount of protein subunit vaccine to be administered to an animal depends on the type and size of animal, the condition being treated, and the nature of the protein, and can be readily determined by one of skill in the art. In fish, for example, if the protein subunit vaccine is to be injected, the amount per injection is preferably between about 0.1 μg and about 1000 μg per 10 g fish; more preferably it is between about 1 μg and about 100 μg per 10 g of fish. For administration by immersion, the concentration of the protein in the aquatic medium is preferably at least about 10 ng/mL; at most it is preferably about 50 μg/mL, preferably it is less than about 1 μg/mL. For oral administration the amount per dose is preferably between about 0.1 μg and about 100 μg per 10 g fish; more preferably it is between about 1 μg and about 10 μg per 10 g of fish. Conveniently, the protein subunit vaccine may include an adjuvant. Further, one or more boosters are preferably administered at time periods subsequent to the initial administration to create a higher level of immune response in the animal. 
         [0027]    In yet another aspect, the vaccine of the invention comprises a fusion protein comprising a carrier polypeptide and said 50 kDa protein of the invention or an analog, fragment, or modification thereof. The 50 kDa protein analog, fragment, or modified protein for use in this aspect of the invention can itself be antigenic or nonantigenic; in embodiments wherein the protein analog, fragment or modified protein is nonantigenic, the carrier polypeptide provides the necessary antigenicity by stimulating the fish&#39;s immune system to react to the fusion protein thereby generating an immune response in an organism, especially fish against  A. hydrophila.  A nonantigenic analog, fragment, or modification of the 50 kDa protein thus functions as a hapten. An example of an antigenic carrier polypeptide is keyhole impet hemocyanim (KLH). Conventional fusion constructs between carriers such as glutathione sulfotransferase (GST) and said 50 kDa protein of the invention or antigenic analog, fragment, or modifications thereof are also included as protein subunit vaccines according to the invention, as are fusions of the 50 kDa protein, analog, fragment or modification and an affinity tag such as a polyhistidine sequence. A fusion construct may be preferred for use as a protein subunit vaccine when the antigenic 50 kDa protein analog, fragment, or modification thereof is small. The invention further provides a method for immunising freshwater fish against  A. hydrophila  by administering to the fish a fusion protein vaccine of the invention. 
         [0028]    A polynucleotide vaccine optionally further comprises a promoter, such as the CMV promoter, operably linked to the coding sequence for the 50 kDa polypeptide or antigenic fragment thereof (e.g., U.S. Pat. No. 5,780,44, Davis). The polynucleotide may be cloned within a vector such as a plasmid. There are numerous plasmids known to those of ordinary skill in the art useful for the production of polynucleotide vaccines. 
         [0029]    Other possible additions to the polynucleotide vaccine constructs include nucleotide sequences encoding cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-12 (IL-12) and co-stimulatory molecules such B7-1, B7-2, CD40. The cytokines can be used in various combinations to fine-tune the response of the animal&#39;s immune system, including both antibody and cytotoxic T lymphocyte responses, to bring out the specific level of response needed to affect the animal&#39;s reproductive system. A polynucleotide vaccine of the invention can also encode a fusion product containing the antigenic polypeptide and a molecule, such as CTLA-4, that directs the fusion product to antigen-presenting cells inside the host. 
         [0030]    Plasmid DNA can also be delivered using attenuated bacteria as delivery system, a method that is suitable for DNA vaccines that are administered orally: Bacteria are transformed with an independently replicating plasmid, which becomes released into the host cell cytoplasm following the death of the attenuated bacterium in the host cell. An alternative approach to delivering the polynucleotide to an animal involves the use of a viral or bacterial vector. Examples of suitable viral vectors include adenovirus, polio virus, pox viruses such as vaccinia, canary pox, and fowl pox, herpes viruses, including catfish herpes virus, adenovirus-associated vector, retroviruses and bacteriophage. Exemplary bacterial vectors include attenuated forms of  Salmonella, Shigella, Edwardsiella ictaluri,  and  Yersinia ruckeri.  Preferably, the polynucleotide is a vector, such as a plasmid, that is capable of autologous expression of the nucleotide sequence encoding said 50 kDa protein or fragment thereof. 
         [0031]    In one embodiment, the vaccine is a DNA vaccine comprising a DNA fragment having a nucleotide sequence that encodes a polypeptide having amino acid sequences shown in  FIG. 8  or  9 , an antigenic analog, fragment, or modified version thereof. 
         [0032]    Polynucleotide-based immunisation induces an immune response to an antigen expressed in vivo from a heterologous polynucleotide fragment introduced into the fish. This method can be advantageous over other methods because heterologous nucleic acid expression may continue for a length of time sufficient to induce a relatively strong and sustained immune response without the need for subsequent “booster” vaccinations, as is common when portions of the protein antigen itself have been injected into the animal. A polynucleotide vaccine comprising a polynucleotide fragment having a nucleotide sequence encoding said 50 kDa protein can be administered to a fish using biolistic bombardment, bath immersion, ingestion or direct injection, as described for example, in U.S. Pat. No. 5,780,448 (Davis), preferably intraperitoneal or intramuscular injection. A preferred method of administration is biolistic bombardment, as with a “gene gun”. A polynucleotide vaccine formulated for oral administration preferably contains DNA encapsulated in a biodegradable polymer. Examples of a suitable biodegradable polymer include chitosan and homo- or co-polyers of polylactic acid and polyglycolic acid. The invention thus further provides a method for immunising freshwater fish or shellfish as against  A. hydrophila  by administering to the fish a polynucleotide vaccine of the invention, preferably a DNA vaccine. 
         [0033]    The amount of polynucleotide vaccine to be administered to an animal depends on the type and size of animal, the condition being treated, and the nature of the polynucleotide, and can be readily determined by one of skill in the art. In fish, for example, if the polynucleotide vaccine is to be injected, the amount per injection is preferably at least about 10 ng; at most it is preferably about 50 μg, more preferably it is less than about 1 μg. If the polynucleotide vaccine is to be administered using a gene gun, the amount per dose is preferably at least about 1 ng; at most it is preferably about 10 μg, more preferably it is less than about 1 μg. For administration by immersion, the concentration of the polynucleotide in the aquatic medium is preferably at least about 10 ng/mL; at most it is preferably about 50 μg/mL, preferably it is less than about 1 μg/mL. For oral administration the amount per dose is preferably at least about 10 μg; at most it is preferably about 10 μg, preferably less than about 1 μg. In some applications, one or more booster administrations of the vaccine at time periods subsequent to the initial administration are useful to create a higher level of immune response in the animal. 
         [0034]    The inventors have observed that serum obtained from infected fish isprotective in passive immunisation experiments, but the activity is serotype-specific. On the other hand, fish that are actively immune following exposure to one serotype may be cross-protected against heterologous strains. Thus, in one embodiment, the vaccine of the invention (whether in the form of a protein vaccine or a polynucleotide vaccine) is monovalent in that it is derived from a particular 50 kDa protein from a particular serotype of  A. hydrophila  and effective to treat or prevent infection of the vaccinated species by that serotype. Preferably, the monovalent vaccine contains at least one antigenic determinant that is shared by the 50 kDa protein of different serotypes, such that is also prevents infection by other  A. hydrophila  of other serotypes, thus offering broad protection. In another embodiment, the vaccine of the invention (whether in the form of a protein vaccine or a polynucleotide vaccine) is a combined vaccine or a multivalent vaccine that prevents infection by other  A. hydrophila  of more than one serotype. The combined or multivalent vaccine can contain or encode, for example, a plurality of serotype-specific 50 kDa S-layer proteins or antigenic portions thereof, derived from multiple serotypes of A. hydrophila, or can contain or encode a synthetic or fusion 50 kDa protein containing multiple antigenic determinants that together generate an immune response against multiple serotypes of A. hydrophila. 
         [0035]    In one embodiment of the vaccine of the invention, the 50 kDa protein or an antigenic portion, analog or modified version thereof may be linked, for example, at its carboxy-terminus to a further component. The further component may serve to facilitate uptake of the 50 kDa protein or an antigenic portion, analog or modified version thereof, or enhance its immuneginicity/processing. For example, the 50 kDa protein or an antigenic portion, analog or modified version thereof may be linked to at least two molecules of the C3d component of complement, using molecular cloning techniques. Preferably, the 50 kDa protein or antigenic portion, analog or modified version thereof is linked to about three molecules of the C3d component of complement. The C3d molecule can be either homologous or heterologous with respect to the species to be vaccinated. Complement genes have been cloned and characterised in salmonids (J. Lambris et al.,  J. Immunol.  151:6123-6134 (1993); J. Sunyer et al.,  Proc. Natl. Acad. Sci USA  93:8546-8551 (1996)). For vaccinations of fish, the 50 kDa protein or antigenic portion, analog or modified version thereof is preferably linked to a salmonid C3d, such as trout C3d or catfish C3d. In the case of a protein subunit vaccine, the recombinant protein is conveniently expressed in bacteria, then administered to fish. The receptor for C3d, namely CD21, is expressed primarily on B cells and the follicular dendritic cells of lymphoid tissues. In the case of a polynucleotide vaccine, a plasmid encoding a fusion protein that incorporates an 50 kDa protein or antigenic portion, analog or modified version thereof, linked at its carboxy-terminus to at least two molecules of the C3d component is administered to the fish. 
         [0036]    The immune-stimulating compositions of the invention may be optionally mixed with excipients or diluents that are pharmaceutically acceptable as carriers and compatible with the active component(s). The term “pharmaceutically acceptable carrier” refers to a carrier(s) that is “acceptable” in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof. Suitable excipients are well known to the person skilled in the art. Examples include; water, saline (e.g. 0.85% sodium chloride; see Ph. Eur. monograph 2001:0062), buffered saline, fish oil with an emulsifier (e.g. a lecithin, Bolec Mont.), inactivant (e.g. formaldehyde; see Ph. Eur. monograph 1997:0193), mineral oils, such as light mineral oils, alhydrogel, aluminium hydroxide. Where used herein, the term “oil adjuvant” to embraces both mineral oils and synthetic oils. A preferred adjuvant is Montanide ISA 711 (SeppicQuai D&#39;Orsay, 75321 Paris, France) which is a manide oleate in an oil suspension. In addition, if desired, the immune-stimulating composition (including vaccine) may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the immune-stimulating composition. 
         [0037]    Thus the present invention also relates to methods for formulation of such proteins to render them suitable for administration by for example immersion or orally via incorporation into fish food. In one embodiment of the invention pertaining to formulation of the vaccine for the immersion vaccination of fish, the polypeptide or nucleic acids may be packaged within a micro-particulate delivery system, which may include, but is not limited to, latex beads, poly(lactide-co-glycolide) microspheres, atelocollagen “minipellets”, bentonite, orporous apatite ceramics including hypoxyapatite (HA) and beta-tricalcium phosphate (TCP). 
         [0038]    For example, for an immersion vaccine, an aqueous suspension is preferred. For an oral vaccine a fish oil and lecithin carrier system may be used. For an injection vaccine Montanide ISA711®, Speic at a ratio of 30:70 may be used. 
         [0039]    A vaccine composition may be administered as a course of a number of discrete doses over a period of time. For example it may be administered over a period of around 2-21 days. 
         [0040]    Vaccination may be repeated at daily, twice-weekly, weekly or monthly intervals. For example a boost vaccination may be administered after the initial dose. For example a boost may be administered at around 4-14 weeks after the vaccination. The initial vaccination and any boost may be carried out using the same or different modes of administration. For example, the initial may be by injection and the boost may be by oral administration. An example regime includes a first vaccination by injection, followed by a course of orally administered boost vaccine, or a booster prior to an expected outbreak. However, it will be appreciated that any suitable route of administration(s) and/or regime(s) may be employed. 
         [0041]    In an example embodiment, the polypeptide or polynucleotide may be diluted to a suitable concentration in an enclosed tank containing water as used for the normal culturing of the relevant fish species and fish fry and the fish/fry are immersed in this solution for a period of say, several hours. The fish may then be returned to their normal culturing conditions. With this practice the polypeptides/polynucleotides may enter the gills or digestive tract of the fish and be engulfed by antigen presenting cells and subsequently induce an immune response. 
         [0042]    In another embodiment, microparticles containing the polypeptides/polynucleotides are incorporated into a typical fish food preparation and fed to fish in place of ordinary feed. In this method the recombinant proteins will enter the digestive tract stimulating an immune response in systemic or gut-associated lymphoid tissues. This method has the advantage of being suitable for use in netted enclosures where sealed tanks are not available. 
         [0043]    Other adjuvants, carriers etc., and modes of administration may be found by referring to Gudding et al (1999) Veterinary Immunology and Immunopathology 72, 203-212. 
         [0044]      Aeromonas hydrophila  is mainly a problem in freshwater, affecting most cultured and wild freshwater fish, although it has been reported in some marine fish, shellfish, amphibians and reptiles. Therefore the species that can be vaccinated with the  A. hydrophila  vaccine is very diverse, and may include animals maintained in aquaculture systems, in public aquaria and by hobbyists, such as the example species listed below. 
         [0045]    Abalone  Haliotis discus hannai;  Adriatic sturgeon  Acipenser naccarii;  African catfish  Clarias bartachus;  African cichlid  Nimbochromis venustus;  Amberjack  Seriola dumerili;  American crayfish  Orconectes limosus, Pacifastacus leniusculus, Procambarus .˜ clarkia;  American plaice  Hippoglossoides platessoides:  Arctic char  Salvelinus alpinus  L.; Atlantic cod  Gadus morhua  L.; Atlantic croaker  Micropogon undulates;  Atlantic menhaden  Brevoortia tyrannus  Latrobe; Atlantic salmon  Salmo salar  L. ; Australian oyster  Saccostrea commercialis;  Ayu  Plecoglossus altivelis;  Balloon molly  Poecilia  spp.; Banana prawn  Penaeus merguiensis;  Barramundi  Lates calcarifer  (Bloch); Bighead carp  Aristichthys nobilis;  Black acara  Cichlasoma bimaculatum;  Black mullet  Mugil cephalus;  Black scraper  Novodon modestus;  Black skirted tetra  Hyphessobrycon  spp.; Blenny  Zoarces viviparous;  Blue fish  Pomatomus saltatrix;  Blue mackerel  Scomber australasicus;  Blue manna crab  Portunus pelagicus;  Blue shrimp  P.  ( Litopenaeus )  stylirostris;  Boney bream  Nematolosa come  (Richardson); Borneo mullet  Liza macrolepis;  Bream  Abramis brama;  Brine shrimp  Artemia;  Brook salmon  Salvelinus fontinalis;  Brook trout  Salvelinus fontinalis  (Mitchill); Brown bullhead  Ictalurus nebulosus  (Lesueur); Brown trout  Salmo trutta;  Brown-spotted grouper  Epinephelus tauvina, E. Coioides;  Bullfrog  Rana catesbeiana;  Burnett salmon  Polydactylus sheridani  (Macleay); Canadian shrimp  Lismata amboiens;  Carp  Cyprinus carpio  L.; Catfish  Clarius batrachus  L.; Caucasian carp  Carassius carassius;  Chanchito  Chichlasoma facetum  (Jenyns); Channel catfish  Ictalurus punctatus  (Rafinesque); Chinook salmon  Oncorhynchus tschawytscha;  Chub  Leuciscus cephalis;  Chum salmon  Oncorhynchus keta  (Walbaum); Cichlid  Oreochromis mossambicus;  Clam  Tapes philippinarum;  Cod  Gadus morhua;  Coho salmon  Oncorhynchus kisutch;  Common carp  Cyprinus carpio  L.; Common snook  Centropomus undecimalis;  Common wolf-fish  Anarhichas lupus;  Coral prawn  Metapenaeopsis  spp.; Crucian carp  Carassius carassius;  Cutthroat trout  Salmo clarki;  Cuttle fish  Sepia officinalis;  Cuvier Plaice  Pleuronectes platessa;  Dab  Limanda limanda;  Dace  Leuciscus leuciscus  L.; Damselfish  Chromis punctipinnis; Pomacentridae, Amphiprion clarkii  (Bennett),  Amblyglyphidodon curacao  (Bloch); Danio  Danio devario;  Discus fish  Symphysodon discus, S. aequifasciatus;  Dolphin fish  Coryphaena hippurus  L.; Eastern freshwater cod  Maccullochella ikei;  Eastern mosquitofish  Gambusia holbrooki  J; Eastern painted turtle  Chrysemys picta picta;  Eel  Anguilla japonica, A. reinhardtii; A. anguilla, A. Rostrata;  European crayfish  Astacus leptodactlus, A. pachypus, A. torrentium, A. astacus, Austropotamobius pallipes;  European sea bass  Dicentrarchus labra;  Fairy shrimp  Branchipus schaefferi  (Fisher),  Chirocephalus diaphanus  (Prevost),  Streptocephalus torvicornis  (Waga); Farmed mussel  Perna perna;  Fathead minnow  Pimephalespromelas;  Flat-tailed mullet  Liza dussumieri  (Valenciennes); Firemouth cichlid  Cichlasoma meeki;  Flounder  Paralichthys olivaceus, Platichthys flesus;  Four bearded rockling  Enchelyopus cimbrius  L.; Freshwater cod (Australian native)  Maccullochella  spp.; Freshwater prawn  Macrobranchium rosenbergii;  Golden shiner  Notemigonus crysoleucas  (Mitchell); Goldfish  Carassius auratus  L; Goldsinny wrasse  Ctenolabrus rupestris;  Gourami (three-spot)  Trichogaster trichopterus;  Grass carp  Ctenopharyngodon idella;  Grayling  Thymallus thymallus  L; Greater weever  Trachinus draco;  Green frog  Rana clamitans;  Green knife fish  Eigemannia virescens;  Green moray eel  Gymnothorax funebris;  Green sturgeon  Acipenser medirostris;  Greenback flounder  Rhombosolea tapirina;  Grouper  Epinephelus guaz, E. coioides;  Guppy  Poecilia reticulata  (Peters),  Lebistes reticulates;  Haddock  Melanogrammus aeglefinus  L.; Halibut  Hippoglossus hippoglossus;  L Herring  Arripis georgianus;  Horse mackerel  Trachurus trachurus;  Iberian toothcarp  Aphanius iberus;  Japanese abalone  Sulculus diversicolor supratexta;  Japanese medaka  Oryzias latipes;  Jewel tetra  Hyphessobrycon callistus  (Boulenger); King prawn  Penaeus latisulcatus;  Knife fish  Gymnotus carapo;  Lake trout  Salmo trutta m lacustris, Salvelinus namaycush  Walbaum; Largemouth bass  Micropterus salmoides;  Leopard frog  Rana pipiens;  Living dace  Tribolodon hakonensis  Gunther; Loach  Misgurnus anguillicaudatus  Cantor; Lobster  Homarus gammarus  L; Local mussel  Mutilus edulis  Long-tom  Tylosurus macleayanus  (Ogilby); Mackerel  Scomberscombrus;  Manila clam  Tapes philippinarum, T. decussatus, Ruditapes philippinarum;  Masu salmon  Oncorhynchus masou;  Menhaden  Brevoortia patronus;  Minnow  Phoxinus phoxinus  L.; Molly  Poecilia velifera  (Regan); Mud crab  Scylla serrata;  Mullet  Mugil cephalus;  Murray cod  Maccullochella peeli:  Mussel  Protothaca jedoensis, Mytilus edulis, Mytilus galloprovincialis;  Mussel (Far-eastern)  Crenomytilus grayanus, Patinopecten yessoensis;  Neon tetra  Paracheirodon innesi, Hyphessobrycon innesi;  North-east Atlantic mackerel  Scomber scombrus;  Northern pike  Esox lucius  L.; Octopus  Octopus vulgaris,  a joubini; One-spot bream  Diplodus sargus;  Ornamental fish  Pterophyllum scalare;  Oscar  Astronotus ocellatus; Apistogramma ocellatus;  Oyster  Ostrea edulis; Crassostreae virginica;  Pacific herring  Clupea harengus palla˜i;&#39;  Pacific oyster  Crassostrea gigas;  Pacific salmon  Oncorhynchus  spp.; Pacific staghorn sculpin  Leptocottus armatus;  Pacific white shrimp  Penaeus vannamei;  Paradise fish  Macropodus opercularis  (L.); Pejerrey  Odonthestes bonariensis;  Perch  Perca fluviatilis;  Pickerel frog  Rana palustris;  Pike  Esox lucius;  Pilchard  Sardinops neopilchardu;  Pink salmon  Oncorhynchus gorbuscha;  Pink snapper  Chrysophrys unicolor;  Pinkfish  Lagodon rhomboids;  Pirarucu  Arapaima gigas;  Pompanos  Trachinotus carolinus  L; Rabbitfish  Siganus rivulatus  (ForsskAl); Rainbow trout  Oncorhynchus mykiss  (Walbaum); Red abalone  Haliotis rufescens;  Red algae  Jainia  spp.; Red claw crayfish  Cherax quadricarinatus  L.; Red drum, Redfish  Sciaenops ocellatus;  Red sea bream  Pagrus major;  Red swamp crawfish  Procambarus clarkia;  Red-eared slider turtle  Chrysemys scripta elegans;  Redtail catfish  Phractocephalus hemiliopterus;  Regan Signal crayfish  Pacifastacus leniusculus;  Roach  Rutilus rutilus  L.; Rohu  Labeo rohita;  Rosy barb  Puntius conchonius;  Rudd  Scardinius erythrophthalmus;  Sablefish  Anoplopoma fimbria  (Pallas); Saltwater crocodile  Crocodylus porosus;  Sand eel  Ammodytes lancea  (Cuvier),  Hyperoplus lanceolatus  (Lesauvege); Sand lance  Ammodytes personatus  Girard; Sand whiting  Sillago ciliata  Cuvier; Saratoga  Scleropages leichardii;  Sardine  Sardinops melanostictus, Sardinops sagnax;  Scallop  Pecten maximus, Argopecten purpuratus;  Scaly mackerel fish  Amblygaster postera;  Sea bream  Pagrus major, Evynnisjaponicus, Sparzrs aurata, Acanthopagrus latus;  Sea catfish  Arius felis;  Sea horse  Hippocampus angustus, H. barbouri, H. whitei, H. kuda;  Sea mullet  Mugil cephalus  L.; Sea trout  Salmo trutta m. trutta  L.; Sea turtle  Chelonia mydas;  Sea-urchin  Paracentrotus lividu;  Sepiolid squid  Euprymna scolopes;  Shorted halibut  Eopsetta grigorjewi;  Shubunkin  Carassius  spp.; Siamese fighting fish  Betta splendens;  Silver black porgy  Acanthopagrus cuvieri, Pyoscelis papua,;  Silver bream  Acanthopagrus butcheri, A. australis  (Owen); Silver bream  Blicca bjoerkna;  Silver carp  Hypophthalmichthys molitrix  Valenciennes; Silver molly  Poecilia  spp.; Silver mullet  Mugil curema  Valenciennes,  Mugil cephalus  L.; Silver perch  Bidyanus bidyanus  (Mitchell); Silver trout  Cynoscion nothus;  Small abalone  Haliotis diversicolor supertexta;  Smallmouth bass  Micropterus dolomieui;  Snakehead fish  Channa striatus; Ophicephalus punctatus, O. striatus;  Snub-nose garfish  Arrhamphus sclerolepsis  (Gunther); Sockeye salmon  Oncorhynchus nerka  (Walbaum); Softshell clam  Mya arenaria;  Sole  Solea solea;  South African abalone  Haliotis midae;  South American side-necked turtle  Podocnemis unifelis;  Spanish mackerel  Scomber japonicus;  Spanner crab  Ranina ranina;  Spiny soft-shelled turtle  Trionyx spinner;  Spot  Leiostomus xanthurus;  Spotted moray eel  Gymnothorax moringa;  Spotted wolf-fish  Anarhichas minor;  Squid  Loligo pealei, Sepiola, T euthoidea;  Starfish  Asterias rubens;  Stingray  Dasyatis pastinaca;  Striped bass  Morone saxatilis  (Walbaum),  M. chrysops;  Striped mullet  Mugil cephalus;  Striped-neck musk turtle  Sternotherus minor peltifer;  Sturgeon  Acipenser naccari;  Sunfish  Mola mola;  Tasmanian lobster  Jasus novaehollandiae;  Tiger Frog  Rana tigrina;  Tilapia  Oreochromis niloticus, O. aurus; Sarotherodon aureus  (Steindachner);  Tilapia nilotica, Tilapia aurea, Tilapia mosambica;  Tom cod  Gadus microgadus;  Tropical shrimp  Stenopus hispidus;  Trout cod  Maccullochella macquariensis;  Turbot  Scophthalmus maximus  L.; Turtle  Dermochelys coriacea; Pseudemis scripta;  Viviparous blenny  Zoarces viviparus;  Western rock lobster  Panulirus cygnus;  White catfish  Ictalurus catus  L.; White clawed crayfish  Austropotamobius pallipes;  White leg shrimp  P.  ( Litopenaeus )  vannamei;  White perch  Roccus americanus, Morone americanus  (Gremlin); Whitefish  Coregonus  spp.; Whitespotted rabbitfish  Siganus canaliculatus;  Whiting  Merlangius merlangus;  Wrasse  Labrus berggylta;  Yellow bass  Morone mississippiensis;  Yellow perch  Perca flavescens;  Yellowfin bream  Acanthopagrus australis  (Owen); Yellowtail  Seriola quinqueradiata, S. lalandi;  Zealand mussel  Perna canaliculus;  Zebra danio  Brachydanio rerio.    
         [0046]    The goal of vaccination against  A. hydrophila  infection is to elicit a population of lymphocytes, which upon subsequent exposure to the parasite proliferate and produce antibodies and/or effector cells specific to the parasite, resulting in protection against lethal infections. A vaccine effective for the prevention of  A. hydrophila  infection in fish is thus one which elicits the production of protective antibodies in a fish exposed to said vaccine. Those protective antibodies will prevent lethal infection of the vaccinated fish upon challenge with  A. hydrophila.  In the present invention, protective antibodies generated in the fish are specific for  A. hydrophila  50 kDa S-layer proteins of the present invention. Fish that can be immunised include ornamental and food fish. 
         [0047]    The present invention further includes monoclonal or polyclonal antibodies, whether derived from fish, rodents, mammals, avians, or other organisms, that bind to the 50 kDa proteins described herein, including antigenic analogs, fragments and modifications thereof. Production and isolation of monoclonal and polyclonal antibodies to a selected polypeptide sequence is routine in the art see for example “Basic methods in Antibody production and characterisation” Howard &amp; Bethell, 2000, Taylor &amp; Francis Ltd. Such antibodies may be used in diagnostic procedures, as well as for passive immunisation. 
         [0048]    Sera from immune fish is known to confer passive immunity against both viral and bacterial pathogens when injected into non-immune fish (Hedrick et al.  Trans. Amer, Fish Soc.  116: 277-281 (1987); Viele et al.  J. Fish Biol.  17:379-386 (1980)) and thus, sera from animals immunised with a polypeptide or polynucleotide according to the present invention, may be of use. 
         [0049]    Additionally, knowledge of the 50 kDa protein nucleotide and amino acid sequences set forth herein also opens up new possibilities for detecting, diagnosing and characterising  A. hydrophila  in fish populations. For example, an oligonucleotide probe or primer based on a conserved region of the 50 kDa protein can be used to detect the presence of the 50 kDa protein in a fish or in water, and an oligonucleotide probe or primer based on a less conserved region can be used to identify a specific A. hydrophila serotype. The invention therefore includes methods for detecting and characterising  A. hydrophila,  for example in aquaculture facilities. 
         [0050]    One aspect of the present invention relates to DNA construct comprising a replicable expression vector and nucleic acid encoding the 50 kDa protein or analog, fragment or modified version thereof. 
         [0051]    Expression vectors for the production of the molecules of the invention include plasmids, phagemids, viruses, bacteriophages, integratable DNA fragments, and other vehicles, which enable the integration of DNA fragments, into the genome of the host. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked genetic control elements that are recognised in a suitable host cell and effect expression of the desired genes. 
         [0052]    These control elements are capable of effecting expression within a suitable host. Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system. Such system typically includes a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of RNA expression, a sequence that encodes a suitable ribosome binding site, RNA splice junctions, sequences that terminate transcription and translation and so forth. Expression vectors usually contain an origin of replication that allows the vector to replicate independently of the host cell. 
         [0053]    A vector may additionally include appropriate restriction sites, antibiotic resistance or other markers for selection of vector containing cells. 
         [0054]    Plasmids are the most commonly used form of vector but other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al. Cloning Vectors: a Laboratory Manual (1985 and supplements), Elsevier, N.Y.; and Rodriquez, et al. (ads.) Vectors: a Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston, Mass (1988), which are incorporated herein by reference. 
         [0055]    In general, such vectors contain in addition specific genes, which are capable of providing phenotypic selection in transformed cells. The use of prokaryotic and eukaryotic viral expression vectors to express the nucleic acid sequences coding for the recombinant proteins of the present invention are also contemplated. 
         [0056]    The vector is introduced into a host cell by methods known to those of skill in the art. Introduction of the vector into the host cell can be accomplished by any method that introduces the construct into the cell, including, for example, calcium phosphate precipitation, microinjection, electroporation or transformation. See, e.g., Current Protocols in. Molecular Biology, Ausuble, F.M., ea., John Wiley &amp; Sons, N.Y. (1989). 
         [0057]    Another aspect relates to a host cell transformed with any one of the nucleic acid constructs of the present invention. Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes. Prokaryotes include gram negative and gram positive organisms, e.g.,  E. coli  and  B. subtilis.  Lower eukaryotes include yeast,  S. cerevisiae  and  Pichia,  and species of the genus  Diclyostelium.    
         [0058]    “Host cell” as used herein refers to cell which can be recombinantly transformed with vectors constructed using recombinant DNA techniques. 
         [0059]    A drug resistance or other selectable marker is intended in part to facilitate the selection of the transformants. Additionally, the presence of a selectable marker, such as drug resistance marker may be of use in keeping contaminating microorganisms from multiplying in the culture medium. Such a pure culture of the transformed host cells would be obtained by culturing the cells under conditions which require the induced phenotype for survival. However, the use of plasmids containing such drug resistance markers when administered directly to a fish to be treated is not desired—plasmids lacking such drug resistance markers are preferred. 
         [0060]    The host cells according to the present invention are capable of producing a biologically active protein or protein fragment of the invention. Such protein .or protein fragment is encoded by the nucleic acid sequence of the invention and is capable of eliciting in an animal protective immunity against a specific adenoviral pathogen. 
         [0061]    A process for the production of the recombinant protein or peptide of the invention is also within the scope of the invention. The process comprises the steps of (a) transforming a host cell with the nucleotide sequence of the invention or transfecting a host cell with a nucleic acid construct of the invention; (b) culturing the cells obtained in (a) under conditions in which expression of the protein takes place; and (c) isolating the expressed recombinant protein or peptide from the cell culture or/and the culture supernatant. 
         [0062]    The polypeptide may be partially purified from the host before being used as a vaccine. Where the polypeptide is secreted from the host cell, the cells may be separated from the media by centrifugation, the cells being pelleted and the media being the supernatant. In such a situation, the supernatant, which contains the secreted polypeptide, may be used directly as a vaccine, or in a vaccine composition. Alternatively, the polypeptide may be partially purified from this supernatant, for example using affinity chromatography. 
         [0063]    The method may further comprise admixing the partially purified polypeptide with another component, such as another polypeptide and/or an adjuvant, diluent or excipient. 
         [0064]    Vaccines may contain bacterial antigens used to control other diseases i.e. vaccine composition may be included within a multivalent vaccine which includes antigens against other diseases of fish. 
         [0065]    In a still further aspect the present invention provides a fish population which has been treated or immunised with a vaccine or composition described elsewhere herein. 
     
    
     
       DETAILED DESCRIPTION 
         [0066]    The present invention will now be further described by way of example and with reference to the Figures which show: 
           [0067]      FIG. 1 : Antibody response of common carp against different isolates of  A. hydrophila  determined by ELISA expressed as an absorbance at 450 nm. All sera were diluted 1:512. 
           [0068]      FIG. 2 : Western blot analysis of different whole cell preparations of  A. hydrophila  against pooled serum from common carp infected with 6 different  A. hydrophila  isolates. Lanes: (1) Standard marker; (2) T4; (3) 98141; (4) Hh; (5) Vds; (6) Catla; (7) C24li; (8) 2D20; (9) 3D14; (10) 2N14; (11) 98140; (12) 98139; (13) B2/12; (14) Fld75; (15) Calf. 
           [0069]      FIG. 3 : Western blot analysis of different preparations of 6  A. hydrophila  isolates screened with serum raised against corresponding  A. hydrophila  isolates in common carp. (A) T4, (B) 98141, (C) Hh, (D) Vds, (E) Catla, (F) C24li. Lanes: (1) Standard marker; (2) WC in vitro; (3) WC in vivo 25 kDa; (4) WC in vivo 100 kDa; (5) OMP in vitro; (6) OMP in vivo 25 kDa; (7) OMP in vivo 100 kDa; (8) ECP in vitro; (9) ECP in vivo 25 kDa; (10) ECP in vivo 100 kDa. 
           [0070]      FIG. 4 : 2D Western blot analysis of whole cell preparation of  A. hydrophila  T4 isolate screened with antibody from common carp infected with  A. hydrophila  T4 isolate. 
           [0071]      FIG. 5 : Cumulative percentage of goldfish mortality in preliminary vaccination trial 
           [0072]      FIG. 6 : MALDI-TOF MS spectrum showing the peptide profiles of 50 kDa band 
           [0073]      FIG. 7 : Nucleic and amino acid sequences of  A. hydrophila  S-layer protein Signal peptide sequences are highlighted (Nucleic acid in green and amino acid in yellow). 
           [0074]      FIG. 8 : Genomic sequences of  A. hydrophila  isolate T4 Bases different to that genome reported for the S-layer protein of  A. hydrophila  isolate TF7 by Thomas and Trust (1995a) have been highlighted. 
           [0075]      FIG. 9 : Amplification of the S-layer gene of  A. hydrophila  isolate T4 shown on a 1% agarose gel. Lanes: (1) Standard marker; (2) S-layer protein gene; (3) purified S-layer protein gene; (4) pQE60 vector carrying S-layer protein gene. 
           [0076]      FIG. 10 : Expression of S-layer protein of  A. hydrophila  with  E. coli  WC protein. (A) 12% SDS-PAGE stained with Coomassie blue, (B) Western blot of protein using an anti-histidine tag antibodies. Lanes: (1) Standard protein marker; (2) WC preparation of recombinant  E. coli  without IPTG induction; (3) WC preparation of recombinant  E. coli  with IPTG induction showing S-layer protein. 
           [0077]      FIG. 11 : Cumulative percentage mortality of carp vaccinated with recombinant S-layer protein and challenged with  A. hydrophila  isolates. (A) T4, (B) Hh, (C) 98140, (D) 98141, (E) Vds, (F) B2/12. 
       
    
    
     MATERIALS AND METHODS  
     Antibody Response of Common Carp to  A. Hydrophila    
       [0078]    Four virulent strains; T4, 98141, Hh, Vds and two avirulent strains; Catla and C24li of  A. hydrophila  were used (Table 1). 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Isolate 
                 Fish species 
                 Country/date 
                 Comments 
               
               
                   
               
             
             
               
                 T4 
                 Rohu 
                 Bangladesh 
                 From an Epizootic 
               
               
                   
                 ( Labeo rohita ) 
                 (1994) 
                 Ulcerative Syndrome 
               
               
                   
                   
                   
                 (EUS) lesion 
               
               
                 98141 
                 Black shark 
                 Ayuthaya Province, Thailand 
                 From a Haemorrhagic 
               
               
                 98140 
                 ( Morulius   
                 (1998) 
                 lesion 
               
               
                 98139 
                   Chrysophekadion ) 
               
               
                 Hh 
                 Hedgehog 
                 Institute of Aquaculture, 
                 — 
               
               
                   
                 ( Erinaceus Europaeus ) 
                 Scotland 
               
               
                 Vds 
                 Catfish 
                 India 
                 From an EUS lesion 
               
               
                   
                 ( Ictulurus puctatus ) 
               
               
                 Catla 
                 Catla 
                 India 
                 From heart blood 
               
               
                   
                 ( Catla catla ) 
               
               
                 C24li 
                 African catfish, 
                 Aquatic Animal 
                 — 
               
               
                   
                 ( Clarias gariepinus ) 
                 Health 
               
               
                   
                   
                 Research Institute, Thailand 
               
               
                 2D20 
                 Frog 
                 Asia 
                 — 
               
               
                 3D14 
                 ( Rana rugulosa ) 
                   
                 — 
               
               
                 2N14 
                   
                   
                 — 
               
               
                 F1d 75 
                   
                   
                 — 
               
               
                 B2/12 
                 unknown 
                 Bangladesh 
                 — 
               
               
                 Calf 
                 unknown 
                 Institute of Aquaculture, 
                 — 
               
               
                   
                   
                 Scotland 
               
               
                   
               
             
          
         
       
     
         [0079]    Common carp (average weight 30 g) were maintained in 6 separate glass tanks. The fish were anesthetized and injected intraperitoneally (IP) with 0.1 ml PBS containing 1×10 6  viable  A. hydrophila.  Each strain was injected into 24 fish and an additional 24 fish were injected with phosphate buffered saline (PBS) as controls. After injection, the fish were supplied with re-circulating water that had been passed through a sedimentation tank, drum filter, biofilter and ultraviolet (UV) radiation. The temperature of the tank water was maintained at 20±1° C. Blood samples were taken 3, 9, 12 and 21 days post-injection and pre-injection bleeds were taken from six fish. Blood was stored overnight at 4° C. and the serum collected by centrifuging at 2000×g for 5 min. The serum was stored at −20° C. until further analysis. 
       Western Blot 
       [0080]    Western blot analysis was performed using fish anti-sera as per the method outlined by Wiens et al. (1990: with modifications). 
       Western Blot Using Common Carp Antibody 
       [0081]    Western blot analysis was carried out for the 14  A. hydrophila  isolates shown in Table 1 using pooled common carp serum raised against six isolates of  A. hydrophila  strains (T4, 98141, Hh, Vds, Catla and C24li). Different preparations of the bacterium (L e. Whole cell (WC), outer membrane protein (OMP) and extracellular products (ECP)) prepared from these 6 isolates of  A. hydrophila  grown either in vitro or in vivo were screened by Western blot with each of the 6 anti-sera raised against different isolates of  A. hydrophila.    
         [0082]    The bacterial preparations described above were subjected to 12% SDS-PAGE and the resolved antigens transferred to nitrocellulose membranes (@60 V for 1 h). The nitrocellulose membranes were blocked using 2% (w/v) casein for 1 h at 20-22° C. The membranes were washed three times (Tris buffered saline containing 0.1% (v/v) Tween-20: TTBS/5 min per wash) and then incubated overnight in the common carp anti-serum diluted 1/10 in Tris buffered saline (TBS). Membranes were again washed and incubated with an anti-carp IgM monoclonal antibody (Aquatic Diagnostics Ltd, Stirling, UK) for 2 h. The membranes were washed and incubated with anti-mouse IgG-HRP (Sigma, Missouri, USA) for 1 h. The blots were developed by adding chromogen and substrate solution (2 ml of 4-chloro-naphthol solution with 10 mls of PBS and 10 μl of H 2 O 2 ) until bands were observed. 
       2D SDS-PAGE Western Blot Using Common Carp Antibody 
       [0083]    The antigenic profile of WC preparation of isolate T4 grown in vitro was screened using 2D SDS-PAGE and Western blotting with anti-serum (raised for T4 isolate) from common carp. 
         [0000]    Assessing the Levels of Protection of a 50 kDa Protein in Goldfish against  A. Hydrophila  Challenge: Preparation of the Antigen. 
         [0084]    Volumes (100 μl) of sample were subjected to 12% SDS PAGE. The gels were run for between 5-6 h at 250 V/130 mA. On completion the 50 kDa band was cut from the gel and finely chopped. These were placed in tubes containing 300 μl SDS-PAGE reservoir buffer a blotting paper disk and a porous polyethylene plug. The tip of the tube was cut and placed into a 1.5 ml centrifuge tube containing 300 μL of 4× SDS-PAGE reservoir buffer. The tubes were then placed into an electroluter and subjected to 50 V at 0.5 mA and a reverse run at 50 V for 5 sec at the end. The eluted protein was collected and the reservoir buffer removed using a 10,000 MW cut-off spin concentrator. The concentration of protein was determined. The presence of the 50 kDa protein was confirmed by SDS-PAGE and Western blot. 
         [0000]    Immunisation of Goldfish with the Electro-Eluted 50 kDa Protein 
         [0085]    For preliminary vaccination, four goldfish weighing around 30-40 g were injected IP with 200 μL of suspension having 12.3 μg of 50 kDa protein in 60 μL of PBS and 140 μL of montanide adjuvant. Another four fish were also injected with PBS to serve as controls. All the fish were challenged with  A. hydrophila  31 days post-vaccination and sacrificed 21 days after challenge as described above. Samples were taken from their kidneys. The relative percentage survival (RPS) was calculated using the following formula (Ellis, 1988). 
         [0000]    
       
         
           
             RPS 
             = 
             
               1 
               - 
               
                 
                   
                     % 
                      
                     
                         
                     
                      
                     vaccinated 
                      
                     
                       
                           
                       
                        
                       
                           
                       
                     
                      
                     mortality 
                   
                   
                     % 
                      
                     
                         
                     
                      
                     control 
                      
                     
                         
                     
                      
                     mortality 
                   
                 
                 × 
                 100 
               
             
           
         
       
     
         [0086]    In addition, two goldfish weighing 30-40 g were injected IP with 200 μL of antigen (i.e. 12.3 μg 50 kDa protein) emulsified with Freund&#39;s complete adjuvant (FCA). Thirty four days later, both fish were re-vaccinated with the same suspension as described above except Freund&#39;s incomplete adjuvant was used in place of FCA. Seventeen days after the booster injection, blood was collected from one fish and the anti-serum collected. 
         [0087]    Three goldfish weighing between 30-40 g were immunised by IP injection with 0.1 ml of goldfish sera raised against the 50 kDa protein electro-eluted from  A. hydrophila,  and 3 fish were injected with control serum collected from non-vaccinated goldfish. After 24 h all the fish were challenged with 0.1 ml of 2.5×10 7  ml −1   . A. hydrophila  T4 isolate in PBS by IP injection, but on the opposite side to the site where they had been injected with the antiserum (LaFrentz, 2003). Kidney samples from fish which died during the experiment and surviving fish at Day 21 post-challenge were streaked on TSA to confirm specific mortality. 
       Sequencing and Identification of the 50 kDa Protein of  A. Hydrophila    
       [0088]    A whole cell preparation of  A. hydrophila  T4 isolate in SDS-PAGE sample buffer was prepared for sequencing and analysis of the 50 kDa protein by MALDI-TOF MS. Samples were in-gel reductively alkylated prior to staining with colloidal Coomassie blue, then digested in 0.1% of n-octyl glucoside/20 mM ammonium bicarbonate plus 12.5 μg ml −1  trypsin, and the sample (1.5 μl) was spotted from the extract (30 μl) after adding an equal volume of acetonitrile for performing MALDI-TOF MS analysis. 
       Recombinant 50 kDa Protein Production 
       [0089]    Recombinant protein was produced in order to have a sufficient quantity of protein for a large scale vaccination trial. All the recombinant protein work was conducted at the Genomic Laboratory, Tokyo University of Marine Sciences and Technology, Japan. 
         [0000]    Polymerase Chain Reaction (PCR) of  A. hydrophila  50 kDa Protein Gene 
         [0090]    Specific primers were designed to amplify the full length of the 50 kDa protein gene based on the sequence data for the S-layer gene of  A. hydrophila  published by Thomas and Trust (1995a: see  FIG. 8 ). Restriction sites Nco I and Bgl II were added to the forward and reverse primers respectively to assist its cloning into the expression vector pQE60. The PCR was run for 32 cycles (95° C./5 min; denaturation for 95° C./30 sec; annealing at 55° C./30 sec; elongation at 72° C./1 min and a final elongation step at 72° C./5 min). The primers used were as follows: Forward: acatgggagttaatctggacactggtgc; reverse: gacttgtggtacttgcgtaagtctaga 
         [0000]    Preparation of PCR Products for Transformation into  E. Coli    
         [0091]    The PCR products were resolved by 1% agarose gel electrophoresis and the DNA was extracted using a DNA purification kit. Digestion of the PCR products and the pQE 60 vector (Qiagen) were carried out overnight at 37° C. Both pQE 60 vectors and PCR products were purified after the digestion process and ligated by mixing 2 μl of vector with 8 μl of PCR products and adding 10 μl ligation high (Cosmo Bio Co Ltd, Tokyo) before incubating it overnight at 16° C. 
         [0000]    Transformation of Vectors Carrying 50 kDa Protein Gene into  E. Coli    
         [0092]      Escherichia coli,  M15 (Quiagen, Tokyo, Japan) was transformed with pQE 60 vectors carrying the amplified 50 kDa protein gene of  A. hydrophila.    
       Expression of the Recombinant 50 kDa Protein in  E. Coli    
       [0093]    The clones containing the 50 kDa protein gene insert identified by PCR, were inoculated into LB broth containing ampicillin (100 μg ml −1 ) and kanamycin (25 μg ml −1 ), and incubated overnight at 37° C. Recombinant protein expression was induced by addition of 1 mM isopropyl-β-thiogalactoside (IPTG) for 4 h. For large scale production, positive clones were cultured in 50 ml of antibiotic supplemented LB broth overnight at 37° C. with vigorous shaking. This culture was transferred to 1 L fresh LB broth and cultured at 37° C. with vigorous shaking. Recombinant protein expression was induced by adding 1 mM IPTG. 
       Sequencing of the  A. Hydrophila  T4 Isolate 50 kDa Protein Gene 
       [0094]    The whole 50 kDa protein gene of  A. hydrophila  isolate T4 was sequenced at the Genomic Laboratory, Tokyo University of Marine Sciences and Technology. 
         [0000]    Vaccination of Common Carp with Recombinant 50 kDa Protein 
         [0095]    Recombinant 50 kDa protein of  A. hydrophila  diluted in PBS was mixed with montanide adjuvant at a ratio of 30:70 (v/v) to a final antigen concentration of 300 μg ml −1 . Buffer (PBS) mixed with the adjuvant was also prepared at the same ratio as the antigen to serve as a negative control. 
         [0096]    One hundred and fifty common carp (30-40 g) were vaccinated by IP injection with 0.1 ml of the vaccine preparation, and another 150 fish were injected with the PBS adjuvant mixture. All the fish were maintained for 35 days in 1×1 m (Diameter×depth) tanks with recirculating water before challenging with six different isolates of  A. hydrophila.    
       Challenge Studies 
       [0097]    Each of the six virulent isolates described above were used to challenge vaccinated fish. Twenty vaccinated and 20 control fish were injected IP with each strain. The concentrations of the bacteria used in the challenge were 1×10 8 , 2×10 7 , 2×10 7 , 5×10 7 , 7.5×10 6  and 2×10 7  bacteria ml −1  for T4, 98140, 98141, Hh, B2/12 and Vds respectively. All 40 fish within each group were placed in separate glass tank (90 cm length×47 cm height×40 cm depth) with aeration and recirculating water. The fish were maintained for 16 days post-challenge and dead fish were removed 3 times a day. Samples from the kidney of dead fish and also from surviving fish at the end of the experiment on Day 16 post-challenge were streaked onto TSA. 
       Statistical Analysis 
       [0098]    The results obtained were statically analysed using Chi-square test for survival, comparing the mortality of vaccinated fish with the control group fish after challenging with bacteria. 
       Results 
       [0099]    Antibody Response of Common Carp Infected with Different Isolates of  A. hydrophila  
 
Antibody Response of Common Carp Infected with  A. Hydrophila  
 
         [0100]    The antibody levels increased after Day 9 and a positive response was observed on Day 12 post-infection with all the isolates, except for isolate 98141. By Day 21 post-infection, this response had increased for all the isolates, with the highest antibody response recorded against isolate T4 ( FIG. 1 ). 
       Western Blot Analysis of Common Carp Serum 
       [0101]    The WC preparations of  A. hydrophila  isolates grown in vitro, screened by Western blot with the anti-sera from infected common carp, exhibited a distribution of bands between 20 and 160 kDa ( FIG. 2 ). Carp antibodies bound to antigens ranging from 30-50 kDa for 3 of the virulent isolates, T4, Hh and B2/12. Except for isolate 2D20, one band was observed at approximately 50 kDa. 
         [0102]    The antibody response against WC, OMP and ECP preparations of  A. hydrophila  grown in vitro and in vivo, showed similar profiles among isolates T4, 98141 and Hh ( FIG. 3 ). With all the virulent isolates (T4, Hh, 98141 and Vds), a band was evident at around 50 kDa in WC and OMP preparations. This band was also present in ECP preparations from in vitro cultured bacteria. 
         [0103]    The OMP preparations from isolate Vds grown in vitro showed 6 bands between 25 and 50 kDa. A band at 50 kDa was observed in WC and OMP preparations of isolate Catla grown in vitro. A band at around 50 kDa was seen in both WC and OMP preparations from isolate C24li grown in vitro and in vivo. Six bands were seen between 35 and 100 kDa with ECP preparations from bacteria grown in MW cut-off tubes but the bands were weakly stained in the case of bacteria grown in the 100 MW cut off tube. 
       2D SDS-PAGE Western Blot 
       [0104]    The 2D Western blot for  A. hydrophila  T4 isolate using the antibody raised against the isolate in common carp expressed three spots at approximately 50 kDa with pI values between 5 and 5.7 ( FIG. 4 ). 
         [0000]    Vaccination and Passive Immunisation of Goldfish with an Electro-Eluted 50 kDa Protein from  A. Hydrophila    
         [0105]    In the vaccination experiment, two control and one vaccinated fish died due to unknown causes before challenging them with  A. hydrophila  isolate T4. The two fish remaining in the control group died on Day one and Day 4 post-challenge ( FIG. 6 ). One fish from the vaccinated group was also sacrificed one week post-challenge as it was suffering from a severe lesion and  A. hydrophila  was isolated from swabs taken from the lesion and kidney of the sacrificed fish. The remaining two fish in the vaccinated group were healthy and sacrificed at the end of the experiment, at 21 days post-challenge. All kidney swabs taken from dead fish were positive for  A. hydrophila  while the samples taken from two vaccinated fish at the end of experiment were negative. Though the numbers of fish used in the experiment were low, the RPS value was 66.7%. 
         [0106]    The fish serum raised against the 50 kDa protein of  A. hydrophila  used to passively immunise fish, had a titre of 1/16. In the trial, one fish from the control group died two days post-infection and the presence of  A. hydrophila  in its kidney was confirmed using an API 20E strip. No other fish died and no kidneys were positive for the bacteria when remaining fish were sampled at the end of the trial on Day 21. 
         [0000]    MALDI-TOF Sequence of the 50 kDa Protein from  A. Hydrophila    
         [0107]    After MALDI-TOF analysis and sequencing, the 50 kDa protein of  A. hydrophila  isolate T4 was identified as a 47.6 kDa S-layer protein ( FIG. 7  and  FIG. 9 ). Six bases were found to be different in the whole S-layer genome of  A. hydrophila  T4 isolate ( FIG. 9 ) compared with the S-layer genome sequence of isolate TF7 reported by Thomas and Trust (1995a:  FIG. 8 ). The amino acid sequence shown in  FIG. 8  was obtained from the NCBI database. 
       Production of a Recombinant Protein for the S-Layer of  A. Hydrophila  Isolate T4 
       [0108]    Bands at 1353 by on the 1% (w/v) agrose gel verified that the amplification of S-layer genomic DNA was successful ( FIG. 10 ). After transformation of vectors into  E. coli  cells, the presence of the S-layer genome in  E. coli  was confirmed by PCR ( FIG. 11   a ) and Western blot ( FIG. 11   b ). 
         [0000]    Standardisation of the Challenge of Common Carp with  A. Hydrophila    
         [0109]    All six strains T4, 98140, 98141, Hh, B2/12 and Vds were passaged twice through common carp and the bacteria were successfully recovered from both passages. During the first passage, no mortalities occurred in any of the groups of fish, while most fish died upon passaging the bacterium a second time with all strains except T4. The values obtained in the preliminary challenge experiment in which the LD 50  dose for each strain was determined are given in Table 2. 
         [0000]                                  TABLE 2                   The LD 50  values of  A. hydrophila  strains for common carp                Strains of  A. hydrophila     LD 50  Value (bacteria ml −1 )                       T4   1 × 10 8             Hh   5 × 10 7             98140   2 × 10 7             98141   2 × 10 7             Vds   2 × 10 7             B2/12   7.5 × 10 6                           
Vaccination of Common Carp with Recombinant S-Layer Protein of  A. Hydrophila  
 
         [0110]    In fish challenged with isolate T4, 75% of control and 10% of vaccinated fish died. A high percentage of mortalities were recorded in control fish challenged with isolate T4 compared with fish challenged with the other isolates of  A. hydrophila.  Fifteen percent of the control group died by the first day post-challenge and 25% had died by Day 2 post-challenge. The levels of mortality decreased to 10% by Day 3 post-challenge and thereafter it varied between 5 and 10% until the mortalities stopped by Day 8 post-challenge. The mortality in the vaccinated group was 5% on Day 1 post-challenge and another 5% had died by Day 5 ( FIG. 12   a ). A relatively high percentage survival (RPS) value (87%) was found with isolate T4 compared with other isolates (Table 3). 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
             
             
               
                   
                   
               
               
                   
                 Total mortality (%) 
                 Relative 
                 P-value 
               
             
          
           
               
                 Strain of 
                 Vaccinated 
                 Control 
                 percentage 
                 (Chi-square 
               
               
                 
                   A. hydrophila 
                 
                 fish 
                 fish 
                 survival (%) 
                 test) 
               
               
                   
               
             
          
           
               
                 T4 
                 10 
                 75 
                 87 
                 0.000 
               
               
                 Hh 
                 10 
                 65 
                 85 
                 0.000 
               
               
                 98140 
                 10 
                 50 
                 80 
                 0.006 
               
               
                 98141 
                 10 
                 40 
                 75 
                 0.028 
               
               
                 Vds 
                 15 
                 40 
                 62.5 
                 0.077 
               
               
                 B2/12 
                 20 
                 45 
                 56 
                 0.091 
               
               
                   
               
             
          
         
       
     
         [0111]    Mortality of 5% was noted in the control group challenged with isolate Hh on Day 1 post-challenge. However, the mortality increased to 20% by Day 2, 15% occurred on Day 5 and 10% on Day 6. The remainder of mortalities (i.e. 15%) were distributed over the period after Day 7. In the vaccinated group, 5% of mortalities were recorded on the first and third day post-challenge ( FIG. 12   b ). The second highest RPS value (85%) in the trial was observed with this isolate. 
         [0112]    Fifty-percentage mortality was seen with the control group challenged with isolate 98140. Thirty percent died in the control group during the first two days post-challenge and remainder died over the course of the experiment (16 days post-challenge). Five percent mortality was recorded in the vaccinated group during the first two days post-challenge, and no further mortalities occurred in this group leading to an 80 RPS value for this isolate ( FIG. 12   c ). 
         [0113]    In the control group challenged with isolate 98141, 25% of mortality occurred over the first two days of the experiment and thereafter 15% mortalities occurred. The mortality with the vaccinated group was similar to that of the mortality recorded with vaccinated group challenged with isolate 98140 ( FIG. 12   d ). An RPS value of 75% was recorded with this isolate. 
         [0114]    The control group challenged with isolate Vds experienced a 10% mortality on Day 1, Day 2 and Day 5 post-challenge, while 5% mortalities occurred on the third and sixth day post-challenge ( FIG. 12   e ). A total of 15% mortality occurred in the vaccinated group distributed over Day 2, 3 and 5 post-challenge. The RPS value with this isolate was 62.5%. 
         [0115]    Percentage mortality in the control group rose to 30% during the first two days after challenging the fish with isolate B2/12. Another 15% mortality occurred in this group over the remainder of the experiment. The highest percentage mortality amongst vaccinated fish was recorded in the group challenged with B2/12. Ten percent mortality was observed in this group on the next day post-challenge and 5% of mortality occurred on the second day and the third day post-challenge ( FIG. 12   f ). The RPS value was low (56%) with this isolate compared to other isolates. 
         [0116]    All the fish that died during the experiment showed the presence of  A. hydrophila  in their kidneys. In contrast,  A. hydrophila  was not cultured from kidney swabs taken from the surviving fish except very few colonies from one fish in the vaccinated group challenged with isolate 98140 and one fish in the control group challenged with isolate 98141. Statistical analysis revealed that survival against isolates T4, 98140, 98141 and Hh were significant in vaccinated fish compared to control fish, while levels of survival were not statistically significant for isolates B2/12 and Vds (Table 3). 
       Discussion 
       [0117]    In this study, common carp were infected with  A. hydrophila  and the anti-sera produced were used to identify immunogenic components of the bacterium. The different  A. hydrophila  isolates examined elicited a variety of responses in common carp, as determined by ELISA. An increase in antibody response against  A. hydrophila  was seen after Day 9 post-infection for all the isolates except one (98141). Antibody response peaked on Day 12 post-infection for two isolates (Hh and Catla) and on Day 21 post-infection for three isolates (T4, Vds and C24li). The antibody response of common carp did not show any differentiation between virulent and avirulent isolates of  A. hydrophila.  This may be due to differences in the ability of the immune system of the host to respond to foreign agents. 
         [0118]    Western blot analysis using the anti-sera produced on Day 21 post-infection, against different strains of  A. hydrophila,  showed differences in the profiles between the isolates. However, when pooled sera (from common carp infected with 6 different isolates) were used to examine the response against the 14 isolates of  A. hydrophila  (described in Table 1), a band at around 50 kDa was observed in all the isolates grown in vitro, except for isolate 2D20. Moreover, bands from 30-50 kDa were stained in the profiles of 3 of the virulent isolates, T4, Hh and B2/12 grown in vitro. A band at around 50 kDa was seen with all the preparations (WC, OMP, ECP) from the virulent isolates grown both in vitro and in vivo compared with avirulent isolates, with the exception of the ECP from bacteria grown in vivo. 
         [0119]    The 2D Western blot analysis of  A. hydrophila  (T4 isolate) WC revealed 3 spots at approximately 50 kDa between 5 and 5.7 μl range when serum raised against T4 isolates was used. The results of the 1D and 2D Western blot analysis suggest that a molecule at approximately 50 kDa (ranging between 47 and 51 kDa) might be one of the major immunogenic components of  A. hydrophila.    
         [0120]    The 50 kDa protein of  A. hydrophila  was considered to be the most immunogenic and most homogenous protein, recognised on each of the  A. hydrophila  isolates examined. The 50 kDa protein conferred protection in goldfish against  A. hydrophila  in the direct immunisation trial. 
         [0121]    After MALDI-TOF MS sequenceing, six bases were found to be different in the whole S-layer genome of  A. hydrophila  T4 isolate ( FIG. 9 ) compared with the S-layer genome sequence of isolate TF7 reported by Thomas and Trust (1995a:  FIG. 8 ). This in turn could result with changes in 4 amino acids in the S-layer protein of  A. hydrophila  isolate T4 compared with S-layer amino acid sequence reported for isolate TF7. 
         [0122]    A recombinant S-layer protein of  A. hydrophila  was produced to confirm the protection efficacy of this protein in common carp against different isolates of  A. hydrophila.  The recombinant S-layer protein proved reactive in Western blot analysis against anti- A. hydrophila  T4 common carp serum and it was used to vaccinate a number of fish. These fish were then challenged with a range of different  A. hydrophila  isolates. High mortality rate was observed both in the vaccinated and control group within two days post-challenge compared with the mortality from Day 3 post-challenge as described in  FIG. 4.8 . The protection elicited by the S-layer protein in vaccinated fish indicates a potential role for this protein in the virulence of  A. hydrophila.    
         [0123]    The S-layer protein antigen of  A. hydrophila  appears to have conferred protection against the different isolates of  A. hydrophila  tested, although the RPS values of carp did vary between the different challenge isolates. 
         [0124]    No mortalities occurred in any of the groups of fish after Day 11 post-challenge in the vaccination trial described in this Chapter. Moreover, no colonies of  A. hydrophila  grew from the kidney swabs taken from surviving fish at the end of experiment except for two fish. This suggests that most of the surviving fish in the control group had cleared the bacterium through their own immune response, as fish can produce an antibody response against different components of bacterium and clear the bacteria in blood circulatory system within seven days post-infection (Leung and Stevenson, 1988b; Chandran et al., 2002b). 
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