Patent application title: NOVEL EDWARDSIELLA ICTALURI E-ICT-VL33 STRAIN, VACCINES THEREOF, AND A METHOD FOR PROTECTING FISHES USING SAID VACCINES
Tsun-Yung Kuo (I-Lan, TW)
Hsu Chung Gabriel Chen (Taipei, TW)
IPC8 Class: AA61K3902FI
Class name: Preparations characterized by special physical form food or edible as carrier for pharmaceutical animal food
Publication date: 2012-02-02
Patent application number: 20120027836
A novel Edwardsiella ictaluri E-ict-VL33 strain, a vaccine derived from
the novel Edwardsiella ictaluri E-ict-VL33 strain, especially in
immersion form and oral form, and a method for protecting fishes from the
infection of Edwardsiella ictaluri, especially catfish, the method
including a primary immersion immunization using immersion vaccine, and
after an appropriate period, boosting by oral vaccination, resulting in a
strong and long-lasting immunity for fishes.
1. A method for improving fish immunity against Edwardsiella ictaluri
comprising: administering an Edwardsiella ictaluri vaccine composition to
fish by immersion for primary immunization; and then administering the
vaccine composition to fish by oral delivery for boost immunization.
2. A method as recited in claim 1, wherein the Edwardsiella ictaluri vaccine composition comprises an Edwardsiella ictaluri E-ict-VL33 strain which is ATCC deposit number PTA-10711, and wherein the Edwardsiella ictaluri vaccine composition is prepared from the inactivation of the Edwardsiella ictaluri E-ict-VL33 strain by inactivation agent.
3. A method as recited in claim 1, wherein before the Edwardsiella ictaluri vaccine composition is administered to fish by oral delivery, the vaccine composition can be spray-coated on the outside of a feed, and then the coated feed can further be sprayed with a oleaginous substance to obtain a feed coated by the vaccine composition.
4. A method as recited in claim 3, wherein the oleaginous substance is at least one selected from the group consisting of: mineral oil, plant oil and animal oil.
CROSS-REFERENCE TO RELATED APPLICATIONS
 The present application is a divisional application of U.S. patent application Ser. No. 12/845,965 filed on Jul. 29, 2010, which is incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The invention relates to a novel Edwardsiella ictaluri E-ict-VL33 strain, vaccines derived therefrom, and a method for protecting fishes using said vaccines.
 2. Brief Description of the Prior Art
 Edwardsiella is a type of small Gram-negative rod bacterium. The host of Edwardsiella includes Catfish, Eel, Tilapia and other warm water fishes. Edwardsiella ictaluri is one member of Edwardsiella. E. ictaluri that can infect catfish, thereby causing Edwardsiella septicemia. Acute Edwardsiella septicemia usually results in typical bacterial septicemia with a high mortality rate for fishes. Chronic Edwardsiella septicemia can result in Hole-in-the-head syndrome, septicemia or, ultimately, death.
 Edwardsiella septicemia occurs worldwide and causes high mortality and considerable economic damage to the catfish industry, especially in Vietnam and the USA. Microbiological infections with E. ictaluri play a major role in catfish (Pangasianodon hypophthalmus) farming in Vietnam [Dung T T, et al., Microbe Drug Res 2008.] and were first observed in the Mekong River Delta in 1999 [Ferguson H W, et al. J Fish Dis 2001; 24:509-13]. E. ictaluri infections are seen in the USA in channel catfish (Ictalurus punctatus) and also in walking catfish in Thailand [Kasornchandra J, et al. J Fish Dis 1987; 10:137-8]. All catfish species are susceptible to E. ictaluri and the disease causes major damage in Basa Tra fish in the Mekong delta and other places where the fish is cultured in Vietnam. Many ways of protecting the fish have been tried but none so far have been very successful.
 The use of antibiotics to counteract bacterial infections has been successful in some instances but not in others, especially because of widespread resistance as a result of extensive and non-controlled use of many different antibiotics either alone or in various combinations. The situation is worsened by the fact that E. ictaluri, and thus resistant E. ictaluri, can survive in the mud of a pond for up to 3 months. Furthermore, the use of antibiotics and chemotherapeutics is discouraged because of both environmental and residual problems, and long-term use of antibiotics is not a sustainable control method for fish diseases. However, preventing and protecting the fish from Edwardsiella septicemia cannot be achieved by using antibiotics which can only control the spreading of Edwardsiella septicemia.
 Vaccination as a preventive measure to control infections with E. ictaluri has been tried in various catfish species. Until now, not a single vaccination or combination of vaccinations has been proven as particularly successful in the field. It has been claimed that an attenuated vaccine is able to help in the control of the disease but, to date, the product has not made major inroads in the field. Besides that, oral delivery of vaccine antigens to fish is the preferred method for several reasons. However the limitations of this method include lack of immune efficacy [Gudding R, et al. Vet Immunol Immunopathol 1999].
 In view of the above-described disadvantages associated with conventional techniques, the inventor had developed a novel Edwardsiella ictaluri E-ict-VL33 strain, vaccines thereof, and a method for protecting fishes using said vaccines.
SUMMARY OF THE INVENTION
 The meaning of the technical and scientific terms as described herein can be clearly understood by the person skilled in the art.
 One object of the invention is to provide a novel Edwardsiella ictaluri E-ict-VL33 strain, which had been identified as a novel strain of E. ictaluri.
 Another object of the invention is to provide a vaccine, which is prepared from the inactivated E. ictaluri E-ict-VL33 strain. Said vaccine can be prepared as an immersion form, oral form or other suitable form.
 Still another object of the invention is to provide a method for improving the immunity of fish against E. ictaluri, and further preventing and protecting fishes from infection of E. ictaluri by using said vaccines.
 In order to achieve the above-described objects of the invention, the invention comprises various aspects as will be discussed below.
 In one aspect of the invention, a novel E. ictaluri E-ict-VL33 strain is to be provided. This strain was isolated from infected tra catfish (Pangasius hypophthalmus). The isolated E-ict-VL33 strain has been cultured and identified as a novel E. ictaluri strain, named E. ictaluri E-ict-VL33, and deposited in the biological resource center ATCC Patent Depository, located at 10801 University Blvd., Manassas, Va. 20110 on Mar. 8, 2010, with an ATCC deposit number PTA-10711 and identification reference by deposit Edwardsiella ictaluri: E-ict-VL33. The bacterium strain of the invention has been used herein is an example, but the invention will not be limited thereto as all kinds of suitable strains are comprised in the invention.
 In another aspect, the invention provides a vaccine composition comprising said novel E. ictaluri strain. In a preferred embodiment, said vaccine composition is a vaccine. The vaccine comprises an effective amount of inactivated E. ictaluri E-ict-VL33 strain and a pharmaceutically acceptable vehicle. It can be prepared in any dosage form suitable for the invention. Said suitable form of the invention includes, but is not limited to, oral-form, immersion-form, injection-form or other suitable forms for the invention.
 An immersion vaccine is obtained by using an inactivating agent to inactivate or kill the E. ictaluri E-ict-VL33 strain (i.e., an inactivation process). The inactivated bacteria is then suspended in a buffer solution. This process allows an immersion vaccine to be obtained.
 An oral vaccine is obtained by using an inactivating agent to inactivate or kill the Edwardsiella ictaluri E-ict-VL33 strain (i.e., an inactivation process). The inactivated bacteria is then suspended in a buffer solution to form a bacterial suspension, and the bacterial suspension is mixed with an adjuvant to form a mixture. The mixture is then homogenously emulsified by a homogenizer with high shear force. This process allows the oral vaccine to be obtained. Said oral vaccine can further be spray coated on the outside of a feed to deliver the oral vaccine to fish. Also, the coated feed can further be sprayed with edible oil, such as plant oil and/or animal oil (in particular, a fish oil), to improve the delivery efficiency of the oral vaccine.
 The term "inactivation" as described herein includes, but is not limited to, treatment with inactivation agent, heat treatment, and other general methods to inactivate or kill the bacteria. The inactivation agent includes, but is not limited to, formaldehyde, binary ethyleneimine (BEI) or other suitable inactivation agents.
 The pharmaceutically acceptable vehicle includes, but is not limited to, solvent, emulsifier, suspending agent, decomposer, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, surfactant, adjuvant or other suitable vehicle.
 The adjuvant includes, but is not limited to, oleaginous adjuvant (such as mineral oil, plant oil, animal oil, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, etc.), aqueous adjuvant (such as aluminum hydroxide), two-phase oleaginous adjuvant (such as water/oil/water form (w/o/w)) etc, and biological (such as adjuvant oligonucleotide and toxoid). The two-phase oleaginous adjuvant comprises a surfactant and an oleaginous substance. The surfactant is selected from the group consisting of: at least one of sorbitol fatty acid ester, the concentrate of sorbitol fatty acid ester and ethylene oxide (or propylene oxide), mannitol fatty acid ester, the concentrate of mannitol fatty acid ester and ethylene oxide (or propylene oxide), modified mannitol fatty acid ester with a hydrophilic group which is selected from the group consisting of: at least one of carboxylic acid, amine, amide, alcohol, polyol, ether and oxide; anhydromannitol fatty acid ester, modified anhydromannitol fatty acid ester with a hydrophilic group which is selected from the group consisting of: at least one of carboxylic acid, amine, amide, alcohol, polyol, ether and oxide; saccharose fatty acid ester, the concentrate of saccharose fatty acid ester and ethylene oxide (or propylene oxide), glycerol fatty acid ester, the concentrate of glycerol fatty acid ester and ethylene oxide (or propylene oxide), the concentrate of fatty acid and ethylene oxide (or propylene oxide), the concentrate of fatty alcohol and ethylene oxide (or propylene oxide), and glycerophospholipid. The oleaginous substance is selected from the group consisting of: at least one of mineral oil, plant oil and animal oil. In a preferred embodiment, said oleaginous substance is animal oil, especially fish oil.
 In addition, the invention also provides a composition comprising the novel E. ictaluri strain. The composition can be applied to various suitable products such as challenge composition containing live bacteria, vaccine containing inactivated bacteria etc.
 In third aspect, the invention provides a method for improving the immunity of fishes against E. ictaluri, further preventing and protecting fishes from infection of E. ictaluri by using said vaccines. According to the following examples, a method comprising a primary immunization by immersion vaccination at first and, after an appropriate time period, a subsequent oral vaccination boosting, can give a protecting effect higher than would be obtained by other vaccination routes. Here, fishes refer to those that are susceptible to Edwardsiella ictaluri infection, in particular, catfishes. The invention also provides the use of Edwardsiella ictaluri E-ict-VL33 in preparation of a vaccine to protect fish from infection of Edwardsiella ictaluri.
 The term "effective amount" refers to the amount of inactivated bacteria used to improve the immunity of fish against E. ictaluri, and then the amount of inactivated bacteria used to prevent and protect the fish from the infection of E. ictaluri or disease derived thereto in further inoculation.
 The term "prevent and protect" is intended to mean that, compared to the immunity of untreated fish, the immunity of treated fish against E. ictaluri is improved and the survival rate of treated fish after infection of E. ictaluri is higher than survival rate of untreated fish. Further inoculation of the amount of inactivated bacteria prevents and protects the fish from the infection of E. ictaluri or disease derived thereto.
 Further scope of the applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
 The present invention will become more fully understood from the detailed description given hereinbelow and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein:
 FIG. 1 shows the morphology of the novel Edwardsiella ictaluri E-ict-VL33 strain under microscope.
 FIG. 2 shows the cumulative mortality (%) obtained after challenge performed at 48 days post-vaccination using an immersion challenge in Groups A, C and E.
 FIG. 3 shows the cumulative mortality (%) obtained after immersion challenge at 121 days post-primary immunization for Groups A, C, E and F.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
 The invention will be illustrated by way of the following examples, but the invention will not be limited thereto.
Isolation and Identification of Edwardsiella ictaluri E-ict-VL33 Strain
 An isolated E-ict-VL33 strain was isolated from infected tra catfish (P. hypophthalmus). Bacterial culture grown in Brain Heart Infusion (BHI, the composition of BHI are shown in Table 1) broth for 18 hours at 25-30° C. was used for experimental infection. Bacteria growth on BHI agar for 48 h at 30° C. was used for bacterial identification.
TABLE-US-00001 TABLE 1 The composition of BHI broth (Difco Laboratories, Sparks, MD) Composition Content Calf Brains, Infusion from 200 g 7.7 g Beef Heart, Infusion from 250 g 9.8 g Proteose Peptone 10.0 g Dextrose 2.0 g Sodium Chloride 5.0 g Disodium Phosphate 2.5 g Add water up to 1 liter Total 1 liter
 The Characteristics of Edwardsiella ictaluri E-ict-VL33 Strain
 FIG. 1 shows that the isolated E-ict-VL33 strain is a rod bacterium. An analysis of Gram staining shows that the isolated E-ict-VL33 strain is a Gram-negative rod bacterium. Referring to Tables 2 to 4, these tables show the growth situation of the isolated E-ict-VL33 strain under different culture conditions such as different temperature, salinity or pH value. "+" represents that the growth of bacteria is slower and contains a lower concentration of bacteria. "++" represents that the bacteria have grown with less precipitation. "+++" represents that the bacteria have grown with higher precipitation. "-" represents that there no growth of bacteria can be observed. In addition, the isolated E-ict-VL33 strain can be cultured in a tryptic soy broth (TSB) or a brain heart infusion (BHI). Furthermore, Table 5 shows the carbohydrate metabolism performance of the isolated E-ict-VL33 strain. "+" represents that the carbohydrate can be metabolized by the isolated E-ict-VL33 strain.
TABLE-US-00002 TABLE 2 Culturing the isolated E-ict-VL33 strain at 28° C., under different pH values. pH value 4 5 6 7 8 9 10 24 hr - + + ++ + + + 48 hr - ++ +++ +++ +++ ++ ++
TABLE-US-00003 TABLE 3 Culturing the isolated E-ict-VL33 strain under different temperatures. Temperature 37° C. 37° C. (AC) 20° C. (AC) 20° C. 28° C. 24 hr - + + - ++ 48 hr - +++ ++ - +++ Note: "AC" stands for acclimatization. In a group labeled "AC," the bacteria is cultured for about 2 day at 28° C., then cultured in a ratio of 1:100 at another temperature. The other groups (i.e., those not labeled "AC") are colonies of bacteria incubated at different temperatures directly.
TABLE-US-00004 TABLE 4 Culturing the isolated E-ict-VL33 strain at 28° C., under different salinity. Salinity 0.5% 1% 1.5% 2% 24 hr + + + - 48 hr ++ ++ ++ -
TABLE-US-00005 TABLE 5 The carbohydrate metabolism performance of the isolated E-ict-VL33 strain. Carbohydrate Performance D-Fructose + Inosine + D-Psicose + Uridine + D-Galactose + α-D-Glucose + D,L-Lactic acid + D-Gluconic acid + N-acetyl-D-Glucosamine + L-Asparagine + Glycerol + D,L-α-Glycerol phosphate + Glycyl-L-Aspartic acid + D-Mannose + D-Glucose-6-Phosphate +
 The Identification of Edwardsiella ictaluri E-ict-VL33 Strain
 After a 16S rDNA analysis, the sequence of 16S rDNA of the isolated E-ict-VL33 strain is shown in SEQ ID No: 1. The result of the 16S rDNA analysis and the sequence alignment of the National Center for Biotechnology Information (NCBI) data bank was higher than 99% identified as E. ictaluri. This demonstrates that the isolated E-ict-VL33 strain is a member of E. ictaluri.
 Because some E. ictaluri strains have a PEI1 plasmid, the inventor used the ORF1 (open reading frame 1) of the PEI1 plasmid (GenBank accession no: AF244083.1) of an E. ictaluri strain as a template to design PCR primers. Sequence of the PEI1 plasmid of the isolated E-ict-VL33 strain was determined by PCR using said primers. After sequencing, a partial sequence of the PEI1 plasmid of the isolated E-ict-VL33 strain is shown in SEQ ID No: 2 (the length of SEQ ID No: 2 is 1876 bp). In a sequence alignment comparing the sequence of SEQ ID NO: 2 with the NCBI data bank, the result indicates that the fragment of the 1st nucleotide (nt) to the 1443rd nt of SEQ ID NO: 2 is similar with the sequence of the PEI1 plasmid (GenBank accession no: AF244083.1) with 99% identity. However, the fragment of the 1444th nt to the 1876th nt of SEQ ID NO: 2 is different from the sequence of the PEI1 plasmid (GenBank accession no: AF244083.1). This indicates that the isolated E-ict-VL33 strain is different from the E. ictaluri strain with the PEI1 plasmid (GenBank accession no: AF244083.1). Although the isolated E-ict-VL33 strain and the E. ictaluri strain with the PEI1 plasmid (GenBank accession no: AF244083.1) are both E. ictaluri, they are different strains according to the sequencing result. Therefore, the isolated E-ict-VL33 strain is a novel E. ictaluri isolated strain.
 The result shows that the isolated Edwardsiella ictaluri E-ict-VL33 strain is identified as a novel Edwardsiella ictaluri strain, and the E-ict-VL33 strain has been deposited in the ATCC on Mar. 8, 2010, with an ATCC deposit number PTA-10711.
Preparation of Vaccines
 Step 1 Cultivation and Collection of the Bacteria
 The bacteria (Edwardsiella ictaluri E-ict-VL33 strain) were cultured in Brain Heart Infusion (BHI) broth at 28° C. for 48 hours, then collected in a bacteria suspension.
 Step 2 Inactivated Processes
 Inactivation of bacteria was done by adding 37% formaldehyde at a final concentration of 0.5% (w/v) to the bacteria suspension obtained in Example 1, and incubating (shaking) the bacteria suspension with formaldehyde at 25° C., 70 rpm for a minimum of 24 hours (preferably 48 hours). The bacteria (Edwardsiella ictaluri E-ict-VL33 strain) have to be verified as being completely inactive by a test. The supernatant of the bacteria suspension containing the formaldehyde was separated by using centrifugation at 9000×g to remove the formaldehyde, then suspending the pellet in a buffer solution (such as distilled water, phosphate buffered saline (PBS)). Finally, the vaccine stock containing inactivated antigen can be obtained. The vaccine stock were stored at +4° C.
Preparation of Immersion Vaccine of Edwardsiella ictaluri E-ict-VL33
 Vaccine stock obtained in example 2 were prepared as immersion vaccine by suspending the vaccine stock in a buffer solution (such as sterile water or PBS). The content of formaldehyde in the immersion vaccine is lower than 0.2% (v/v).
Preparation of Oral Vaccine of Edwardsiella ictaluri E-ict-VL33
 Vaccine stock obtained in example 2 were prepared as a 400 liter oral vaccine by mixing 300 liters of vaccine stock (at least comprising 1.65×1015 cfu bacteria), 4 liters of surfactant (polysorbate 80), and 96 liters of fish oil. The antigen of E. ictaluri was coated with a two phase oleaginous adjuvant in a w/o/w form by homogenously emulsifying the mixture with a homogenizer with high shear force (10,000 rpm). The oral vaccine can then be obtained. The 400 liter oral vaccine is composed of 24% (v/v) fish oil, 1% (v/v) surfactant, and 3.85×108 cfu/ml inactivated bacteria. The content of formaldehyde in the oral vaccine is lower than 0.2% (v/v).
 Use of the Oral Vaccine
 The invention also provides the application of the oral vaccine obtained in example 4. The oral vaccine can further be spray coated on the outside of commercially available pelleted feed at 2% (volume/weight). Also, the coated pellet can further be sprayed with squid oil at 0.1% (v/w). The feed can be AQUAXCEL 7434 pelleted feed or other commercially available feed.
Efficiency of the Edwardsiella ictaluri E-ict-VL33 Inactivated Vaccine
 5-1. Efficiency Experiment
 Healthy, non-infected, catfish (P. hypophthalmus), locally referred to as Tra fish, were vaccinated using immersion or oral vaccination or combinations of these vaccinations. The various vaccinations or vaccination combinations and challenge preparation were divided into various experiments for the (sub) groups. The various vaccinations or vaccination combinations and challenges were performed in at least 3 different experiments.
 Experiment 1: Set Up the Challenge Model
 A total of about 1500 fish were used to set up the challenge model to be used in the trial.
 Bath challenge: Fifty fish per tank in three parallel tanks per dose (with 4 doses total) were challenged by pouring bacteria grown in BHI broth into water to give a final concentration of 5.5×103 to 5.5×106 colony forming units (5.5×103, 5.5×104, 5.5×105 and 5.5×106) of E. ictaluri E-ict-VL33 strain per ml of water. Exposure to the challenge dose lasted for 30 min. Strong aeration was supplied to the water in the tanks during the challenge. The control groups were immersed in clean aerated water. Mortalities were monitored for 14 days.
 Injection challenge: Before injecting, fish were anaesthetized with 0.2% MS222 in the water. Fish were injected intraperitoneally with 0.1 ml of a bacterial dilution at 4 different bacterial concentrations ranging from 5.5×103 to 5.5×106 colony forming units (10-fold steps, 5.5×103, 5.5×104, 5.5×105 and 5.5×106) of E. ictaluri per ml or sterile saline water for control. Mortalities were monitored for 14 days.
 From both experiments, moribund and freshly dead fish were submitted for bacterial isolation from the liver, spleen and kidney. Surviving fish at the end of monitoring period were checked for bacterial infection.
 Experiment 2: Immersion/Oral Immunization Studies
 Combined immersion/oral immunization studies are summarized in Table 6. The groups included Group A (immersion-prime) with immunization by immersion only (day 1) and challenge at 48 and 121 days after primary vaccination, Group C (imm-oral boost-1) with combined immersion (day 1) and oral boost by days 8 to 21, and Group F (imm-oral boost-2) boosted a second time through experimental days 101 to 107. Group E (oral-prime) was given primary immunization at days 8 to 21 by the oral route and challenged at 48 and 121 days after primary vaccination. Non-vaccinated control groups were challenged at 48 and 121 days.
 Table 6 outlines how the immersion and oral vaccination of the various fish was done and which groups were challenged using the method established in Experiment 1.
TABLE-US-00006 Challenge Challenge Number at day 48 at day 121 of primary first second (50 fish per (40 fish per Group replicates vaccination boost boost parallel) parallel) A 3 Immersion-prime -- -- Yes -- 1 Immersion-prime -- -- -- Yes C 3 Immersion-prime Oral -- Yes -- (day 8-21) 1 Immersion-prime Oral -- -- Yes (day 8-21) F 1 Immersion-prime Oral Oral -- Yes (day 8-21) (day 101-107) E 3 Oral -- -- Yes -- (day 8-21) 1 Oral -- -- -- Yes (day 8-21) Ct 3 Non-vaccinated Yes -- controls 1 Non-vaccinated -- Yes controls Note: Ct represents the control group and "--" represents the group without performing the treatment.
 The process of immersion vaccination, oral vaccination, and immersion challenge are described as follow:
 Immersion vaccination: The immersion vaccine consisted of a sterile water-based, killed bacterial suspension of 5.0×109 bacteria per ml Immersion vaccination was performed by immersing 1200 fish in 2 L of vaccine stock diluted in 18 L (5.56×108 bacteria per ml final concentration) of clean water for 1 min with strong aeration.
 Oral vaccination: The outside of feed pellets was spray coated by the oral vaccine obtained in Example 4 (in a w/o/w form, containing 3.85×108 cfu/ml) at 2% (volume/weight). The coated pellets were then sprayed with squid oil at 0.1% (v/w). The coated feed pellets were prepared daily and used within 1 to 2 days after preparation and fed to satiation. The feed can be WOOSUNGVINA Co. J3, J4 feed or other commercially available feed.
 Immersion challenge: The immersion challenge was performed in 96 liter tanks containing 70 liters of clean water. At the first challenge (day 48), 40 or 50 fish (number of fish depending on the experiment) from each tank were transferred to a bucket containing 10 liter of clean water and then bacteria grown in BHI broth was poured into the water to given concentrations of 7.6×106 or 4.3×106 bacteria/ml of water for Experiments 2 and 3, respectively. Exposure to the challenge dose lasted for 1 h. At the second challenge (121 days), the same immersion method was applied with a concentration of 8.1×106 bacteria/ml of water for Experiments 2. The non-challenged controls were immersed in clean aerated water. After challenge the fish were observed for 14 days.
 Statistical Analysis
 Fisher's exact test was used to analyze differences between groups at end-point. A P-value below 0.05 was considered to represent significant differences between groups/treatments.
 5-2. Results
 Experiment 1
 In this experiment, challenge by immersion was compared with challenge by injection. For immersion challenge the end-point mortality varied from 1.3% (±1.15 SD) at 5.5×103 cfu/ml to 66% (±8.5 SD) at 5.5×106 cfu/ml. For the injection challenge, it ranged from 93 (±1.5 SD) to 99.3% (±0.6 SD) end-point mortality over the dose range tested. Based on these results, immersion challenge was used for assessing vaccination efficacy and it was considered that the immersion dose of >106 gave sufficient mortality, i.e. more than 60% control mortality.
 Experiment 2
 Referring to FIG. 2 and Table 7, immersion/oral immunization studies (Experiment 2) showed a cumulative mortality in the non-vaccinated controls of 87% by day 48. In Group A (immersion-prime) the average cumulative mortality was 65% (±3.1 S.D.) (p<0.02), while in Group E (oral prime) average, cumulative mortality was 74%±3.5 (p>0.1), and in Group C (imm-oral boost-1) average cumulative mortality was 42%±4.0, (p<0.001), giving RPS values of 25, 15, and 52, respectively. The results indicate clearly that the fish given a combination of immersion and oral boost (C group) were much better protected than the fish in the other groups (A or E group).
Relative Percent Survival(RPS)=(1-(% mortality in vaccinated fish/% mortality in control))×100.
TABLE-US-00007 TABLE 7 Cumulated mortality of the vaccinated and control fish in experiment 2 at first challenge (experiment day 48; 27 days post completion of oral boost). Vaccination method/group Immersion Immersion/oral Days after (only) (1x) Oral (only) Control challenge A1 A2 A3 C1 C2 C3 E1 E2 E3 Ct1 Ct2 Ct3 1 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 6 1 0 0 0 0 1 1 2 0 0 0 0 7 4 0 3 2 1 4 3 3 4 7 6 3 8 9 6 7 4 3 4 6 10 6 10 10 6 9 11 7 4 6 5 6 10 7 11 6 12 9 10 5 8 5 5 4 5 8 4 12 7 6 12 11 3 4 9 1 3 2 5 4 1 7 4 5 12 1 5 5 1 1 0 2 5 4 1 2 7 13 0 1 0 2 1 1 3 0 0 3 2 0 14 0 0 0 0 1 0 0 0 0 2 2 2 Total mortality 34 31 33 21 19 23 38 35 38 43 44 44 No. of fish/tank 50 50 50 50 50 50 50 50 50 50 50 50 Mortality (%) 68 62 66 42 38 46 76 70 76 86 88 88 Average mortality 65% 42% 74% 87% RPS 25 52 15 --
 Referring to FIG. 3 and Table 8, the second challenge was performed at Experimental day 121 and fish were given a challenge dose of 8.1×106 cfu/ml of water. Cumulated mortality in the different vaccine groups and the control fish is shown in Table 10. At experimental day 121 the cumulative mortality in the controls was 90%. In Group A (immersion-prime) cumulative mortality was 80% (RPS=11, p=0.26), while Group E (oral prime) showed a mortality of 82% (RPS=9, p=0.388). In Group C (imm-oral boost-1) mortality was 64% (RPS=29, p=0.0037) while in Group F (imm-oral boost-2) cumulative mortality was 48% (RPS=47, p=0.0001). From the statistical evaluation only a combined vaccination of immersion/oral gives significant protection over the controls and single immunization procedures.
TABLE-US-00008 TABLE 8 Cumulated mortality of the vaccinated and control fish in experiment 2 at second challenge (experiment day 121). Vaccination regimes/Group Immersion/oral Immersion/ (2 boost; 21 Days after Immersion oral (21 d) Oral Controls day and 107*) challenge A C E Ct F 1 0 0 0 0 0 2 0 0 0 0 0 3 0 0 0 0 0 4 0 0 1 1 0 5 1 2 3 3 1 6 3 3 2 1 2 7 3 3 5 4 2 8 8 5 8 13 5 9 10 7 10 9 7 10 5 5 5 4 4 11 5 5 4 3 2 12 4 2 1 3 1 13 1 0 2 2 0 14 0 0 0 2 0 Total 40 32 41 45 24 mortality Total 50 50 50 50 50 number of fish/tank Mortality 80 64 82 90 48 (%) RPS 11 29 9 -- 47% *the numbers indicate the experiment days that the oral boost feeding ended
 The results show that a single immersion or oral immunization regime confers low protective immunity at long term post immunization. A combination of immersion and one oral boost is superior to the immersion (only) method. A second boost initiated 80 days after the first boost and completed 21 days before challenge resulted in an increased level of protection and gives a RPS=47 at a control mortality of 90%.
 Together these findings show that a primary immunization with combined immersion and oral delivery induces a strong and long-lasting immunity Immunized fish benefit significantly from a second boost via the oral route.
 In summary, the invention provides:
 1. A novel Edwardsiella ictaluri E-ict-VL33 strain.
 2. Vaccines derived from the novel Edwardsiella ictaluri E-ict-VL33 strain.
 3. A method for improving the immunity of fishes against Edwardsiella ictaluri, further preventing and promoting fishes from the infection of Edwardsiella ictaluri.
 Many changes and modifications in the above described embodiment of the invention can, of course, be carried out without departing from the scopes thereof. Accordingly, to promote the progress in science and the useful arts, the invention is disclosed and is intended to be limited only by the scope of the appended claims.
<160> NUMBER OF SEQ ID NOS: 2
<210> SEQ ID NO 1
<211> LENGTH: 1410
<212> TYPE: DNA
<213> ORGANISM: Edwardsiella ictaluri E-ict-VL33
<223> OTHER INFORMATION: 16S rDNA of Edwardsiella ictaluri
<400> SEQUENCE: 1
gcggacgggt gagtaatgtc tggggatctg cctgatggag ggggataact actggaaacg 60
gtagctaata ccgcataacg tcgcaagacc aaagtggggg accttcgggc ctcatgccat 120
cagatgaacc cagatgggat tagctagtag gtggggtaat ggctcaccta ggcgacgatc 180
cctagctggt ctgagaggat gaccagccac actggaactg agacacggtc cagactccta 240
cgggaggcag cagtggggaa tattgcacaa tgggcgcaag cctgatgcag ccatgccgcg 300
tgtatgaaga aggccttcgg gttgtaaagt actttcagta gggaggaagg tgtgagcgtt 360
aatagcgttc acaattgacg ttacctacag aagaagcacc ggctaactcc gtgccagcag 420
ccgcggtaat acggagggtg caagcgttaa tcggaattac tgggcgtaaa gcgcacgcag 480
gcggtttgtt aagttggatg tgaaatcccc gggcttaacc tgggaactgc atccaagact 540
ggcaagctag agtctcgtag agggaggtag aattccaggt gtagcggtga aatgcgtaga 600
gatctggagg aataccggtg gcgaaggcgg cctcctggac gaagactgac gctcaggtgc 660
gaaagcgtgg ggagcaaaca ggattagata ccctggtagt ccacgctgta aacgatgtcg 720
atttggaggt tgtgcccttg aggcgtggct tccgaagcta acgcgttaaa tcgaccgcct 780
ggggagtacg gccgcaaggt taaaactcaa atgaattgac gggggcccgc acaagcggtg 840
gagcatgtgg tttaattcga tgcaacgcga agaaccttac ctactcttga catccagcga 900
atcctgtaga gatacgggag tgccttcggg aacgctgaga caggtgctgc atggctgtcg 960
tcagctcgtg ttgtgaaatg ttgggttaag tcccgcaacg agcgcaaccc ttatcctttg 1020
ttgccagcgg ttaggccggg aactcaaagg agactgccag tgataaactg gaggaaggtg 1080
gggatgacgt caagtcatca tggcccttac gagtagggct acacacgtgc tacaatggcg 1140
tatacaaaga gaagcgacct cgcgagagca agcggacctc ataaagtacg tcgtagtccg 1200
gattggagtc tgcaactcga ctccatgaag tcggaatcgc tagtaatcgt ggatcagaat 1260
gccacggtga atacgttccc gggccttgta cacaccgccc gtcacaccat gggagtgggt 1320
tgcaaaagaa gtaggtagct taaccttcgg gagggcgctt accactttgt gattcatgac 1380
tggggtgaag tcgtaacaag tacgtggact 1410
<210> SEQ ID NO 2
<211> LENGTH: 1876
<212> TYPE: DNA
<213> ORGANISM: Edwardsiella ictaluri E-ict-VL33
<223> OTHER INFORMATION: partial sequence of the plasmid pEI1 of
Edwardsiella ictaluri E-ict-VL33
<400> SEQUENCE: 1
atgccattgc atgtcggcac cgggcatatc cccgccacca tcagtaataa ccgcattcat 60
cgcatcgcca gcgctaatac cccaccggag atgagcgtgc gggagaatat tcaggagttt 120
ttctgctcaa cacataagtc agaggccctt aactgccttt gggaaatttg tcatcctcca 180
gtcggaacta cacgggacgc tgtagcgggt agatttgagc agctcaagag actcgcctat 240
ccgggatacg agaataatat ccagtctggc cgaaatggag ataacagctt ctgtattttg 300
gatgaagacg gacaggaaat gttatctgta atccttggtg atgccgatac gtataccgtt 360
atctatccgg gtggcgagat gacacatgac ctcagccttt ccgcaccaca aggagatgag 420
ggtctcctga tattaggtgc ttccgacacc gtgggaccac aaacagaggg agagtataaa 480
gctgcctggt cagcgtggga aagggctgcg ccaccagaag aggcagaaga ccgagccaag 540
acagtacggg agctgcgtca ctgcctgcga tataacaccc ggctggatgt ctctaacacc 600
aaactaacca gtttgccgcc tctgccgacc ggactgaaga ggctggatgt ctctggtact 660
ggactgacca gcctgccgcc actccatcca aactgcagag gctggatatc tctaacactc 720
ctatgccaag gctaccgtca ctgccctcca gactgcagag actggatgtc tctaacaccc 780
acctggccaa ccttccggaa atcatcgcca atttatcgca gaggacaacg gtaagactgc 840
agagtaactc cctgtcagaa cgtacacttc agatgatcaa taaacccggc tatcagggac 900
ccaggatagt tgatatggca acatcttcta atcatcagga aaccagagcc ctgcacttgg 960
cggtggcgga ctggctgacg ccggcggaag cggacgagga ggcttccgct aaaagatggt 1020
gggctatcgg gaaggaagac gatgccgccg ccttcagcac tttcctcgac cggctgaaaa 1080
agacgaaaaa taatcaggca accgattttc acgtgcagat agccagctgg attagccagc 1140
tggcgcagga tgatgagcta agagccaaaa cctttgacat ggcgacagag tcaacctcaa 1200
gctgtgagga cagggtcacg ctggcaatga gcaatatgta gagtgtgcaa ctggtccata 1260
atgctgaaaa agggaaattc gaccgtgata tcccggggct cgtgtcagcg gggcgtgaaa 1320
tgttccgcct agaacaacta gatctaattg cccgtgagaa ggtaaaaaag ctacgctttt 1380
tcgatgaaat tgagataaaa aaatggctat cagaacaaac tgaaagaatc acttgaacta 1440
acctccaaac ccgtcggccg gcgcattgag cagattccgc ggtcaaggcc atcgacgttg 1500
ggtcatctgg ggggaaccga gcgcgggact gggagcatcc tctagcgttt gaggtggccc 1560
cagagggcgc ccgtcctcgc gtatcaggga tatagagtct cgcgtcacaa ttgacttcat 1620
agatggagga gcagtcggtg gcgttctcca gcagcgggga atcccccaac agctgggtca 1680
gcagcagata tagggggttt ctctgcagat ggaaacgaca cctcagtatc ctccatttat 1740
ggaaatcatt tccccaccag aacctatatc cgctatagtc aggaacgcgg gatctccggg 1800
gccatctcaa agttgagaaa acccgagtcc cctccgtttt ctccgagcgc acaacagacc 1860
accccggctc gaaaaa 1876
Patent applications by Tsun-Yung Kuo, I-Lan TW
Patent applications in class Animal food
Patent applications in all subclasses Animal food