In ovo methods for utilizing live Edwardsiella ictaluri against enteric septicemia in channel catfish

Methods for the safe and effective live in ovo vaccination of eyed eggs of catfish against enteric septicemia of catfish (ESC) were created through the use of rifampicin resistant native E. ictaluri isolates; these including rifampicin-resistant strain ATCC 202058 of Edwardsiella ictaluri originally isolated from the walking catfish Clarius batrachus from Thailand. Single immersion exposure of eyed eggs of catfish stimulated strong acquired immunity against many isolates of E. ictaluri without the need for booster immunization.

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 novel vaccine methods against ESC which provides 
effective in ovo protection; this being related 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 in ovo methods that are safe and effective for the 
control of ESC in catfish. The vaccines comprise one or more rifampicin 
(3-[4-methylpiperazinyl-iminomethyl]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 in ovo live vaccination against ESC in catfish. 
It is also an object of this invention to provide for in ovo vaccination of 
catfish utilizing both monovalent and polyvalent vaccines against ESC. 
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.'s B-21909, B-21910, and B-21911 
respectively. 
DETAILED DESCRIPTION OF THE INVENTION 
"Vaccine" 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 "revertant" 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 term "in ovo" is intended to refer to the treatment of eyed eggs of 
catfish prior to hatch. 
The term "eyed egg" refers to that stage in egg development where the eye 
of the fish embryo is visible through the egg wall. This typically occurs 
in catfish 4-5 days after egg laying, and 3-4 days prior to hatch, where 
the water temperature is 78.degree. F. 
The starting material for use in preparing the vaccines of the invention is 
any attenuated Edwardsiella ictaluri bacterium such as the ATCC 202058 
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 "effective immunization dosage" 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. Typically, vaccination is carried out by exposing channel 
catfish eyed eggs by immersion in water containing about 1.times.10.sup.5 
CFU/ml of attenuated Edwardsiella ictaluri for ten minutes at a density of 
about 50 grams of eyed fish eggs per liter of water and vaccine and a 
temperature of about 25.degree. 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.times.10.sup.4 to about 
1.times.10.sup.8 CFU/ml of immersion medium. Useable vaccination times are 
seen to range from about 2 minutes 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 20.degree. C. to about 28.degree. C., most 
preferably from about 22.degree. C. to about 26.degree. C. Concentrations 
of eyed fish eggs treated in the inoculation medium typically range from 
about 50 grams to about 100 grams per liter of immersive innoculant. 
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 ATCC 202058 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 .mu.g/ml instead of 200 .mu.g/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.degree. 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'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.degree. 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.degree. 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 .mu.g/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.degree. 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 .mu.g/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 .mu.g/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 .mu.g/ml was created. 
Edwardsiella ictaluri isolate ATCC 202058 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 .mu.g/ml rifampicin 
for 33 passages. The resultant mutant, designated ATCC 202058, is 
differentiated from the parent microorganism because it can survive and 
reproduce without negative effect on a media containing 320 .mu.g/ml 
rifampicin. Biochemical characteristics of the E. ictaluri ATCC 202058 are 
identical to E. ictaluri as described in Bergey's Manual of Determinative 
Bacteriology (Holt et al.,1994), herein incorporated by reference. 
EXAMPLE 2 
Eyed channel catfish eggs totaling 500 grams in weight were vaccinated by 
immersion with about 1.times.10.sup.5 CFU/ml ATCC 202058 vaccine for ten 
minutes at a density of about 50 grams of eyed eggs/L in 24-26.degree. C. 
water. The fish subsequently hatched from the immunized eggs were kept in 
1500L fiberglass tanks supplied with recirculating well water at 
26.degree. C. with a flow rate of 0.5L/minute. Fish were fed daily a 
commercial catfish ration amounting to 4% of their body weight. Fish 
utilized in the experiment ranged in size from about 3 grams to about 5 
grams and ranged from about 33 to about 36 days of age. Fish were observed 
daily for mortalities, abnormal behavior, and to monitor for any signs of 
ESC. In the experiment, consisting of a trial lasting from 33 to 36 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 
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PROTECTION AGAINST ENTERIC SEPTICEMIA OF CATFISH (ESC) 
AFTER IMMERSION VACCINATION OF EYED CHANNEL CATFISH 
EGGS WITH EDWARDSIELLA ICTALURI VACCINE (ATCC 202058) 
# Dead fish/ Relative percent 
# challenged 
% mortality survival (RPS) 
______________________________________ 
Control.sup.a 
Tank 1 24/30 80.0 
Tank 2 6/30 20.0 
Tank 3 11/30 36.6 
mean 45.5 
Vaccinated 
Tank 1 5/30 16.6 
Tank 2 0/30 0.0 
Tank 3 0/30 0.0 
mean 5.5 87.9 
______________________________________ 
.sup.a nonvaccinated eggs 
Eyed egg mass (500 g) vaccinated with ATCC 202058 modified live E. ictaluri 
vaccine in 9.5 liters of water; 33 to 36 day old fish were challenged with 
E. ictaluri (AL-93-75) by immersion. Eggs were vaccinated in water and 
vaccine for 10 minutes. No fish died after vaccination. 
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## 
At sixteen days post challenge (DPC) the relative percent survival was 
87.9%. Five of 90 vaccinates died after challenge. In this study, 
mortality in an equivalent group of 90 untreated controls was 41 of 90 or 
45.5%. 
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.