Abstract:
Safe and effective live vaccines against enteric septicemia of catfish (ESC) were created through the induction of rifampicin resistance in native E. ictaluri isolates; these including rifampicin-resistant strain (RE-33) of Edwardsiella ictaluri originally isolated from the walking catfish Clarius batrachus from Thailand. Single immersion exposure of catfish fry stimulated strong acquired immunity against many isolates of E. ictaluri without the need for booster immunization.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), is a Gram-negative bacterium that is highly infectious for the channel catfish Ictalurus punctatus. This pathogen has been isolated from channel catfish in most areas of the United States where this species is cultured and from walking catfish in Thailand [Kasornchandra et al., J. Fish Dis. 10:137-138(1987). Losses in fingerlings to market-size fish account for about 50% of the total disease losses to catfish producers. Formalin-killed bacterial vaccines have not been successful due to their provoking only a humoral response. This is due to Edwardsiella ictaluri being an intracellular pathogen, with acquired immunity in fish surviving ESC being more cellular than humoral. 
     This invention relates to a novel vaccine against ESC which does in fact provide superior protection over existing commercial vaccines due to its ability to stimulate cellular-immunity and protection. 
     2. Description of the Prior Art 
     It has been estimated that ESC is responsible for annual losses of 20 to 30 million dollars in the catfish industry. Control of E. ictaluri has been attempted by feeding antibiotic medicated feed. This expensive practice has been ineffective because sick fish do not eat and antibiotic resistance to oxytetracycline and ormethoprim-sulfamethoxine, the approved drugs for use on food fish, has been observed. 
     Based on the success of disease control by bacterin immunization of salmonids, bacterins were developed against ESC. An E. ictaluri bacterin (ESC vaccine, ALPHARMA, formerly BIOMED, Inc., 1720-130th Ave. N.W., Bellevue, Wash., USA) was produced and marketed with a provisional license for immersion vaccination of channel catfish against ESC. An oral E. ictaluri bacterin (Escogen, Aqua Health, Ltd., Charlottetown, PEI, Canada) was produced, marketed and licensed for oral vaccination of channel catfish against ESC. Thune et al. [J. Appl. Aquaculture 3, 11-23 (1994)] teach that vaccination of channel catfish (Ictalurus punctatus) with E. ictaluri bacterins has not resulted in acquired immunity of long duration. Nusbaum and Morrison [J. Aquat. Animal Health 8, 146-149 (1996)] also demonstrated that killed E. ictaluri was not entering the fish and suggested that this affected vaccine efficacy. Edwardsiella ictaluri has been described as a facultative intracellular pathogen of channel catfish [Miyazaki et al.; J. Fish Dis. 8, 389-392 (1985)]. Because of the nature of E. ictaluri as an intracellular pathogen, it is not surprising that killed vaccines have not been successful. Strictly controlled live vaccination of channel catfish with low concentrations of unmodified isolates of E. ictaluri by bath immersion was found to stimulate protective immunity, specific antibodies by Klesius et al. [Journal of Aquatic Animal Health 7, 205-210 (1995)]. Protective immunity to B. abortus in cattle was demonstrated by Montaraz et al. [Infect. Immun. 53, 245-251 (1986)] and Schurig et al. [Vet. Micro. 28, 171-188 (1991)] using vaccines based on modified live rifampicin-resistant Brucella species. 
     SUMMARY OF THE INVENTION 
     We have now discovered a means for the creation of novel live vaccines that are safe and effective for the control of ESC in catfish. The vaccines comprise one or more rifampicin (3-[4-methylpiperazinyliminomethyl]rifamycin SV) (Sigma Chemical Company, St. Louis, Mo.) resistant mutant isolates of E. ictaluri, created by multiple passaging of native isolates on increasing concentrations of rifampicin. These vaccines are effective in providing long lasting acquired immunity in channel catfish to ESC. 
     In accordance with this discovery, it is an object of the invention to provide a novel, highly protective, live vaccine against ESC in catfish. 
     It is also an object of this invention to provide both monovalent and polyvalent vaccines against ESC that are more efficacious than those presently in commercial use. 
     It is another object to provide an attenuated Edwardsiella ictaluri vaccine that is safe and provides long lasting acquired immunity in channel catfish to enteric septicemia of catfish. 
     It is a further object of this invention to improve the viability and productivity of catfish, and to reduce economic losses in the catfish industry caused by ESC. 
     Other objects and advantages of the invention will become readily apparent from the ensuing description. 
     Deposit of Biological Material 
     Rifampicin-resistant attenuated Edwardsiella ictaluri isolate EILO RE-33, was deposited on Nov. 26, 1997, under the provisions of the Budapest Treaty with the American Type Culture Collection located at 10801 University Boulevard, Manassas, Va. 20110-2209, and has been assigned Accession No. ATCC 202058. 
     Rifampicin-resistant attenuated Edwardsiella ictaluri isolates RA-75-34, RS-629-34, and RS-694-34 were deposited on Jan. 12, 1998 under the provisions of the Budapest Treaty in the Agricultural Research Service Culture Collection located at 1815 North University Street, Peoria, Ill. 61604, and have been assigned Deposit No.&#39;s B-21909, B-21910, and B-21911 respectively. 
     DETAILED DESCRIPTION OF THE INVENTION 
     &#34;Vaccine&#34; is defined herein in its broad sense to refer to any type of biological agent in an administratable form capable of stimulating a protective immune response in an animal inoculated with the vaccine. For purposes of this invention, the vaccine may comprise one or more live attenuated isolates of Edwardsiella ictaluri having the characteristic of rifampicin-resistance. 
     The starting material for use in preparing the vaccines of the invention may be any isolate of Edwardsiella ictaluri. Serial passage of the isolate of Edwardsiella ictaluri over increasing concentrations of rifampicin produces strains with an attenuated pathogenicity efficacious for the preparation of live vaccines. The methodology for attenuation by serial passage is well known and documented in the art as exemplified by Schurig et al. [Vet. Micro. 28, 171-188 (1991)], hereby incorporated by reference, who created vaccines based on modified live rifampicin-resistant Brucella species. 
     The term &#34;revertant&#34; is intended to refer to a subculture of an attenuated bacterium, the subculture being characterized by increased virulence and increased in vivo replication as compared to the attenuated form. The term is used herein generically to encompass both true revertants and apparent revertants, the latter being derived from an existing bacterial population. 
     The starting material for use in preparing the vaccines of the invention is any attenuated Edwardsiella ictaluri bacterium such as the EILO RE-33 bacterium reported supra. The attenuation achieved by high-level serial passage in culture on increasing concentrations of rifampicin virtually eliminates the pathogenicity of the bacterium toward channel catfish. The native strain of Edwardsiella ictaluri should be passaged a sufficient number of times such that in its new attenuated form it no longer possesses the ability of causing the disease state known as enteric septicemia in catfish. The efficacy of the monovalent vaccine against challenge by certain strains of native Edwardsiella ictaluri is however not universal. The vaccinal efficacy can be enhanced by combining multiple attenuated strains of Edwardsiella ictaluri into bivalent or polyvalent vaccines. 
     Vaccination, while being accomplishable by injection or through oral ingestion, is most efficiently done by means of aqueous immersion. The bacterial agent is prepared for administration by formulation in an effective immunization dosage with an acceptable carrier or diluent, such as water. The expression &#34;effective immunization dosage&#34; is defined as being that amount which will induce immunity in a catfish against challenge by a virulent strain of ESC. Immunity is considered as having been induced in a population of catfish when the level of protection for the population is significantly higher than that of an unvaccinated control group. One measure of the level of protection is the protective index (PI), which is calculated as the ESC in unvaccinated, ESC bacteria challenged controls minus the ESC in vaccinated, ESC bacteria challenged groups, and the difference divided by the percent ESC in unvaccinated, ESC bacteria challenged controls, with the result multiplied by 100. Typically, vaccination is carried out by exposing channel catfish by immersion in water containing about 1×10 5  CFU/ml of attenuated Edwardsiella ictaluri for two minutes at a density of about 50 fish/L and a temperature of about 25° C. These parameters may be varied as desired such that a sufficient level of vaccination is acquired without induction of excessive loss. Useable concentrations of Edwardsiella ictaluri are considered to range from about 5×10 4  to about 1×10 8  CFU/ml of immersion medium. Useable vaccination times are seen to range from about 1 minute to about 60 minutes, preferably from about 2 minutes to about 10 minutes. Temperature of the inoculation media may range within the physiologically acceptable limits of catfish, preferably from about 18° C. to about 28° C., most preferably from about 22° C. to about 26° C. Concentrations of fish treated in the inoculation medium typically range from about 50 to about 100 fish/L, but, in the alternative, be determined on a weight basis and range from about 0.5 to about 2.5 kg/L. The vaccine can be effectively administered anytime after the catfish attains immunocompetence, which is at about the second day post-hatch. 
     Appropriate adjuvants as known in the art may also be included in the vaccine formulation. In many cases, the vaccinal efficacy can be enhanced by combining the different strains of attenuated Edwardsiella ictaluri into bivalent or polyvalent vaccines. 
    
    
     The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention which is defined by the claims. 
     EXAMPLE 1 
     The procedure used to produce the RE-33 vaccine was modified from that described in Shurig et al. (1991; Vet Micro 28, 171-188), hereby incorporated by reference, by using a lower initial concentration of rifampicin and ending at 320 μg/ml instead of 200 μg/ml and by omitting the penicillin step. 
     Process of Developing Resistant Mutants of Edwardsiella ictaluri 
     Brain heart infusion agar plates for the cultivation of Edwardsiella ictaluri were made according to the procedure of Difco (Difco, Inc., Detroit, Mich.). 37 g of brain heart infusion media and 17 g of agar are added to one liter of distilled water. The media and agar were heated until dissolution. The media was then autoclaved at 121-124° C. for 15 minutes, the media was then poured into sterile petri dishes (15 ml per dish) and allowed to solidify before refrigeration. 
     Native isolates of Edwardsiella ictaluri were obtained from sick catfish or previously obtained lyophilized stocks. Isolates of E. ictaluri were then identified by standard biochemical tests as set forth in Bergey&#39;s Manual of Determinative Bacteriology prior to use in rifampicin resistant E. ictaluri. After identification, the process of forming rifampicin resistant isolates of Edwardsiella ictaluri was begun. Rifampicin supplemented brain heart infusion agar plates were prepared as follows: Brain heart infusion agar was made as described above and sterilized at 121-124° C. for 15 minutes. After sterilization, the correct amount of rifampicin was added to the media prior to its solidification and 15 ml of the resulting mixture was poured into separate petri dishes and allowed to solidify prior to refrigerated storage. Initial cultures of the native isolates of Edwardsiella ictaluri were grown on brain heart infusion agar plates which were incubated at 20-25° C. for 24-48 hours or until 1-2 mm translucent colonies were observed. A single E. ictaluri colony was then picked with a sterile inoculating loop and streaked onto a rifampicin supplemented brain heart infusion agar plate containing the correct concentration of the antibiotic. For the initial passage, rifampicin was present in the brain heart infusion agar at a concentration of 5 μg/ml. The rifampicin supplemented brain heart infusion agar which was streaked with the aforementioned native isolate of E. ictaluri was then incubated for 24-48 hours at 20-25° C. and observed for bacterial growth. Single colonies of E. ictaluri which grew on the rifampicin supplemented media were then picked and placed onto the next concentration of rifampicin (10 μg/ml) brain heart infusion agar plates. If growth occurs, a single colony is harvested and placed on an agar media containing the next higher concentration of rifampicin (20 μg/ml). If the harvested colony failed to grow, it was repeatedly passed on a media containing the last concentration of rifampicin at which growth successfully occurred, before being placed on the next higher concentration of rifampicin containing media. This process was repeated until a colony capable of growing on a media containing a rifampicin concentration of 320 μg/ml was created. 
     Edwardsiella ictaluri isolate EILO was passaged on increasing concentrations of rifampicin (3-[4-methylpiperazinyl-iminomethyl]rifamycin SV) (Sigma Chemical Company, St. Louis, Mo.) supplemented brain heart infusion (BHI) agar to a final concentration of 320 μg/ml rifampicin for 33 passages. The resultant mutant, designated RE-33, is differentiated from the parent microorganism because it can survive and reproduce without negative effect on a media containing 320 μg/ml rifampicin. Biochemical characteristics of the E. ictaluri EILO RE-33 are identical to E. ictaluri as described in Bergey&#39;s Manual of Determinative Bacteriology (Holt et al., 1994), herein incorporated by reference. 
     EXAMPLE 2 
     Lipopolysaccharide (LPS) was extracted as described by Schurig et al. (1991, ibid.). Bacterial growth from a 250 ml BHI broth culture of both the parent EILO and RE-33 was harvested by centrifugation. The cell pellet was then suspended in 25 ml of 10 mM Tris, pH 8.0 and bacteria were killed by addition of an equal volume of acetone, and stirred overnight. Cells were centrifuged at 13,000×g for 5 minutes at 4° C. and then washed with 100 ml of sterile distilled water. The bacteria were then resuspended in 45 ml of sterile distilled water and then 55 ml of phenol was added (incubated at 68° C. for 40 minutes). The mixture was then centrifuged at 17,000×g at 4° C. for 10 minutes. The phenol layer was removed and the process repeated three times. The phenol phases were pooled and washed with hot distilled water (about 66° C.), and centrifuged at 17,000×g at 4° C. for 10 minutes and the resulting pellet harvested in 10 ml sterile distilled water. This was then frozen at -70° C. and subsequently lyophilized. Samples of phenol extracted LPS from E. ictaluri EILO and RE-33 were treated as described by Schurig et al. (1991; ibid.) prior to electrophoresis. SDS-PAGE electrophoresis was then carried out using the Phast system (Pharmatech-Biotech, Uppsala, Sweden) with 10-15% gradient gels. The gels were then silver stained to examine for the presence or absence of the O-side chain of LPS. Silver stained SDS-PAGE profiles of lipopolysaccharide extracted from E. ictaluri RE-33 indicated that the O-chain was not present as compared to the parent E. ictaluri EILO lipopolysaccharide. 
     EXAMPLE 3 
     A total of 16,460 channel catfish, blue catfish (Ictalurus furcatus), and blue catfish x channel catfish free of E. ictaluri were vaccinated by immersion with about 1×10 5  CFU/ml RE-33 vaccine for two minutes at a density of about 50 fish/L in 24-26° C. water. Immunized fish were kept in 1500 L fiberglass tanks supplied with recirculating or well water at 18 or 26° C. with a flow rate of 0.5 L/minute. Fish were fed daily a commercial catfish ration at 4% of their body weight. Fish utilized in the experiment ranged in size from about 10 grams to about 50 grams and ranged from about 3 to about 9 months of age. Fish were observed daily for mortalities, abnormal behavior, and to monitor for any signs of ESC. In the experiment, comprising 10 separate runs lasting from 14 to 120 days post vaccination, no signs of ESC or mortality were seen in any of the fish. The results are presented below in Table I. 
     
                       TABLE I______________________________________SAFETY OF VACCINE DOSE OF EDWARDSIELLA ICTALURI  RE-33 IN CHANNEL CATFISH, BLUE CATFISH AND BLUE  CATFISH X CHANNEL CATFISH VACCINATES                             Number Alive/Day Post Signs of  Experi-  Species or Numbers Vaccination ECS or  ment Date Strains Vaccinated (DPV) Mortality______________________________________1      7/96    Marion    60      60/14  None  2 10/96  USDA  125 125/14 None  3 10/96  USDA 1050 1050/60 None  4 11/96  USDA  525 525/30 None  5 12/96  USDA 1100  1100/120 None  6 1/97 USDA 1100 1100/90 None  7 5/97 USDA 3500 3500/90 None  8 5/97 Norris X 3000 3000/90 None    USDA  9 5/97 Blue X 3000 3000/90 None    USDA  10 5/97 Blue 3000 3000/90 None    Total 16460  16460 None______________________________________ 
    
     EXAMPLE 4 
     Channel catfish (125 USDA and 60 Marion strain) were vaccinated following the protocols of EXAMPLE 3 at 200 times the normal vaccine dose (2×10 7  /ml) for 30 times longer exposure (1 hour) to determine the safety of the vaccine. Results of experimental challenge are presented as relative percent survival (RPS) as described by Amend (1981; Dev. Biol. Stand. 49, 447-454), herein incorporated by reference. RPS is calculated according to the following formula: ##EQU1## 
     A positive effect by vaccination is a RPS greater than 50%. 
     At fourteen days post vaccination (DPV) the relative percent survival was 94.7%. No fish died after the vaccination. In this study, mortality in an equivalent group of 125 untreated controls was 92%. 
     EXAMPLE 5 
     Twenty catfish (blue x channel catfish) were injected intra peritoneally (IP) with 50 μl of a 24 hour culture of RE-33 which was isolated from channel catfish after vaccination. Reversion to virulence did not occur in any of the twenty fish tested. No mortality or signs of ESC were observed in the fish for 35 days after injection. 
     EXAMPLE 6 
     Five serial passages of E. ictaluri RE-33 were conducted in five groups of ten channel catfish to examine reversion to virulence. E. ictaluri RE-33 was obtained from a vaccinated channel catfish and grown in BHI broth for 24 hours before use. The first group of ten fish was vaccinated at the normal dose and time. Two to three days following vaccination, two to three fish were euthanized and cultured for E. ictaluri RE-33. After a pure culture was obtained, the process was repeated five times. Fifty channel catfish were immersed in 2 ml/L RE-33 culture which had been streaked on 320 μg/ml rifampicin supplemented BHI agar plates to non-rifampicin supplemented plates for 16 passages. Reversion to virulence did not occur. The fish used in this experiment were held in the laboratory without signs of ESC or adverse behavior for at least 20 days. Edwardsiella ictaluri RE-33 was not isolated from five fish sampled 12 days after injection. No mortality or signs of ESC were observed for 35 days in the 50 fish utilized in the test. 
     EXAMPLE 7 
     Induction of protective immunity by E. ictaluri RE-33 was examined in relation to 12 different E. ictaluri isolates obtained from diseased channel catfish throughout the Southeast and the parent isolate from Thailand. These are listed below in Table II. 
     
                       TABLE II______________________________________EDWARDSIELLA ICTALURI ISOLATES USED IN EXPERIMENTS  Isolate     Source              Location______________________________________AL-93-75  channel catfish with ESC                         Alabama  AL-95-58 channel catfish with ESC Alabama  AL-96-25 channel catfish with ESC Alabama  ATCC-33202 American Type Culture Collection Georgia  S94-629 channel catfish with ESC Mississippi  S94-649 channel catfish with ESC Mississippi  S94-707 channel catfish with ESC Mississippi  S94-827 channel catfish with ESC Mississippi  S94-873 channel catfish with ESC Mississippi  S94-1017 channel catfish with ESC Mississippi  S94-1051 channel catfish with ESC Mississippi  S94-1034 channel catfish with ESC Mississippi  EILO walking catfish Thailand______________________________________ 
    
     Groups of either 60 or 125 channel catfish were vaccinated with E. ictaluri RE-33 according to the protocols set forth in Example 3. At 14 days post vaccination these fish were challenged with the 13 E. ictaluri isolates at concentrations of 2×10 7  /ml for one hour. Controls were immersed in brain heart infusion broth only. Relative percent survivals were greater than or equal to 50% at 14 DPV (days post vaccination) for 8 of the 13 isolates tested (see Table III). 
     
                                           TABLE III__________________________________________________________________________PROTECTION AGAINST ENTERIC SEPTICEMIA OF CATFISH (ESC)  AFTER IMMERSION VACCINATION.sup.1 OF CHANNEL CATFISH  WITH EDWARDSIELLA ICTALURI RE-33 VACCINE                      Relative                            Percent   Days Post Number of E. ictaluri Percent Mortality   Vaccination Fish Challenge.sup.2 Survival in  Experiment (DPV) Vaccinated Isolate (RPS).sup.3 Controls__________________________________________________________________________1      14     60    EILO.sup.4                      51.7  100.0  2 14 60 AL-93-75 98.3 100.0  3 14 125 AL-93-75 96.6 94.6  4 14 60 AL-93-75 96.8 94.4  5 84 60 AL-93-75 93.9 26.4*  6 105 60 AL-93-75 78.9 79.3  7 14 60 S94-873 54.0 68.3  8 14 60 S94-1017 71.7 100.0  9 14 60 S94-1051 50.0 100.0  10 14 60 AL-96-25 50.4 96.7  11 14 60 S94-827 47.0 96.7  12 14 60 S94-1034 27.0 98.3  13 14 60 ATCC-33202 6.7 100.0  14 14 125 AL-95-58 78.4 40.8*  15 14 125 S94-629 53.3 24.0*  16 14 60 S94-694 18.3 100.0  17 42 60 S94-694 96.0 55.0*  18 63 60 S94-694 100.0 55.0*  19 14 60 S94-707 29.0 91.7  20 42 60 S94-707 93.0 70.0Total         1395  13 isolates                      Mean Protection = 64.6%__________________________________________________________________________ .sup.1 Immersion vaccination with 1 × 10.sup.5 E. ictaluri RE33/ml for 2 minutes at a density of about 50 fish per L of water. .sup.2 Challenge with E. ictaluri isolates at 1 to 2 × 10.sup.7 /ml for 1 h described by Klesius and Sealey (1995) and Shoemaker and Klesius (1997). .sup.3 Relative percent survival (RPS) as determined by Amend (1981). RPS&#39;s ≦50% are considered protection by vaccination (Amend, 1981). .sup.4 Parent of RE33 vaccine strain. *Data do not fit Amend&#39;s criteria of 60% mortality in controls. 
    
     By increasing the time after vaccination, protection was demonstrated as having been developed for two more of the isolates (S94-694 and S94-707). 
     EXAMPLE 8 
     Induction of protective immunity by E. ictaluri mutants RA-75-34, RS-629-34 and RS-694-34 were examined in relation to native E. ictaluri isolate AL-93-75 obtained from channel catfish in Alabama. Groups of 75 channel catfish were vaccinated with E. ictaluri mutants according to the protocols set forth in Example 3. At 14 days post vaccination these fish were challenged with the native E. ictaluri isolate at a concentration of 2×10 7  /ml for one hour. Controls were immersed in brain heart infusion broth only. Relative percent survivals were greater than or equal to 50% at 14 DPV for vaccinated groups tested (see Table IV). 
     
                       TABLE IV______________________________________EFFICACY OF RIFAMPICIN RESISTANT E. ICTALURI MUTANTS  RA-75-34, RS-629-34 AND RS-694-34 IN CHANNEL CATFISH  CHALLENGED WITH E. ICTALURI                  Fish Dead Due    Relative   Number to ESC after Percent Percent  Treatment.sup.1 of Fish Challenge Mortality Survival______________________________________Non-vaccinated     75       35         46.67   --  controls  RA-75-34.sup.2 75 2 2.67 94.3  RS-629-34 75 7 9.33 80.0  RS-694-34 75 17 22.67 51.4______________________________________ .sup.1 All fish were alive and free of signs of ESC for 21 days after vaccination. .sup.2 Fish were vaccinated at about 1 × 10.sup.7 CFU / ml of each mutant. 
    
     It is understood that the foregoing detailed description is given merely by way of illustration and that modification and variations may be made therein without departing from the spirit and scope of the invention.