Patent Application: US-33376794-A

Abstract:
a method is provided for the enhanced elaboration of leukotoxin from f . necrophorum , and subsequent production of an inactivated leukotoxoid ruminant animal vaccine against f . necrophorum infection and consequent liver abscesses and / or foot rot in such animals . the method involves forming a culture of f . necrophorum bacteria in growth media , allowing the bacteria to grow therein and to simultaneously elaborate leukotoxin in a supernate ; the culturing is preferably carried out at a temperature of from about 35 °- 41 ° c ., a ph of from about 6 . 5 - 8 , and for a period of from about 4 - 9 hours . at the end of the culturing , bacterial growth and leukotoxin elaboration are terminated , preferably by separating the leukotoxin supernate , whereupon the vaccine is produced by inactivation of at least the supernate .

Description:
the following examples describe the preferred techniques for the elaboration of f . necrophorum leukotoxin , as well as the subsequent production of leukotoxoid vaccine . it is to be understood , however , that these examples are presented by way of illustration only , and nothing therein should be taken as a limitation upon the overall scope of the invention . the following example describes a series of tests undertaken to determine the conditions for maximal elaboration of leukotoxin from various strains of f . necrophorum . full citations for referenced articles appear at the end of the description . twenty - four f . necrophorum strains ( 9 biotype a and 15 biotype b ) previously isolated from bovine liver abscesses ( lechtenberg et al ., 1988 ) and strains 2159 and 5076 ( biotype a ) and 5111 ( biotype b ) obtained from dr . john berg , university of missouri , columbia , were used . bacteria were grown in prereduced ( 0 . 05 % cysteine hcl ), anaerobically sterilized , bhi broth ( difco laboratories , detroit , mich .) at 39 ° c . the anaerobic techniques used for preparation and dispensing of medium , inoculation , and sample removal were according to holdeman et al . ( 1977 ). to minimize variation , the log - phase ( determined spectro - photometrically ) bacterial growth was used as inoculum in all experiments . colony counts of f . necrophorum were determined in anaerobic bhi agar by the hungate roll tube method ( hungate , 1969 ). growth was monitored spectro - photometrically at a 660 initially and at 1 hour intervals until maximum absorbance was recorded . specific growth rate was calculated according to koch ( 1981 ). culture supernate for leukotoxin assay was obtained by centrifugation at 15 , 000 × g for 30 minutes at 4 ° c . the supernate was filtered through a 0 . 2 - μm membrane filter ( micron separations , inc ., westborough , mass .) and mixed with an equal volume of phosphate - buffered saline ( pbs ; ph 7 . 4 ). samples were stored at - 70 ° c . and assayed for leukotoxicity . leukotoxicity was quantitated by a tetrazolium ( mtt )- dye ( 3 -[ 4 , 5 - dimethylthiazoyl - 2yl ]- 2 , 5 - diphenyltetrazolium bromide ) reduction assay with bovine polymorphonuclear neutrophil ( pmn ) leukocytes as target cells ( vega et al ., 1987 ). blood pmn cells were isolated as described by reddy et al . ( 1990 ). briefly , blood was collected from the jugular vein into heparinized tubes and centrifuged at 1 , 500 × g for 10 minutes at 40 ° c . plasma , buffy coat layer , and one - third of red blood cells ( rbc ) layer were discarded . the sediment containing pmn was treated with 0 . 83 %- ammonium hcl ( in 10 mm tris buffer , ph 7 . 5 ) for 1 minute to lyse remaining rbc , then was mixed with 20 ml of rpmi - 1640 ( gibco laboratory , grand island , n . y .). after centrifugation at 500 × g for 10 minutes , the pellet was collected and retreated with ammonium hcl . isolated pmn were suspended in rpmi - 1640 medium , supplemented with fetal bovine serum ( 5 %), l - glutamine ( 1 mm ), penicillin ( 5 , 000 u / ml ) and streptomycin ( 5 , 000 μg / ml ), at a final concentration of 2 . 5 × 10 6 pmn cells / ml . cell concentration and viability (& gt ; 97 %) were determined by the trypan blue dye exclusion method . one hundred μl of pmn suspension was dispensed into each well of a 96 - well , flat - bottomed , cell culture microtitration plate and incubated overnight in 5 % co 2 humidified air atmosphere at 37 ° c . to allow adherence of neutrophils . after incubation , the medium was aspirated with a vacuum pump from each well to remove nonadherent cells and replenished with 100 μl of rpmi - 1640 medium . an equal volume of serially diluted culture supernates was added into each well containing the neutrophils . the mixtures were incubated for 1 hour followed by addition of 20 μl of 0 . 5 % ( w / v ) mtt - dye ( sigma chemical co ., st . louis , mo .) per well . after 3 hours incubation in 5 % of co 2 at 37 ° c ., the formazan concentration representing viability was determined by measuring absorbance in an elisa reader with dual wavelength ( 570 nm as test wavelength and 650 nm as reference ). the leukotoxicity , expressed in percentage of cell death , was calculated as follows : the titer of leukotoxin was calculated as the reciprocal of the culture supernate dilution causing 10 % loss in viability of leukocytes . to evaluate the time course of leukotoxin production , f . necrophorum biotype a strains 21 and 25 and biotype b strains 16 and 35 were grown in 100 ml of anaerobic bhi broth . five ml aliquots were collected at 0 , 2 , 4 , 6 , 8 , 10 , 12 , 16 and 29 hours for colony counts and toxin assay . because leukotoxin production peaked at late - log phase ( 6 - 8 hours ) of growth of both biotypes , subsequent samples for leukotoxin assay were obtained at late - log phase ( 6 - 8 hours ), unless otherwise specified . to compare leukotoxin production between biotypes , 27 f . necrophorum strains ( 11 biotype a and 16 biotype b ) were grown in anaerobic bhi broth . culture supernates from late - log phase were assayed for leukotoxicity . effect of culture medium , ph and incubation temperature on growth and leukotoxin production f . necrophorum biotype a , strain 25 was grown under different cultural conditions . in order to select a suitable commercial medium that would support maximal leukotoxin production , bacteria were grown in bhi , eugon , and liver infusion broths ( difco ) at 39 ° c . all growth media were prereduced with cysteine hydrochloride and sterilized anaerobically . the inocula were obtained by growing the bacteria in the same medium . to evaluate the effect of medium ph on growth and leukotoxin production , bacteria were grown in anaerobic bhi broth with ph of 6 . 7 , 7 . 3 , 7 . 7 , and 8 . 2 at 39 ° c . medium ph was adjusted by adding 1 n naoh or 1 n hcl . to compare the growth and toxin production under various incubation temperatures , bacteria in anaerobic bhi broths ( ph 7 . 7 ) were incubated 30 , 35 , 39 and 43 ° c . in all instances , samples for leukotoxin assay were obtained at 6 - 8 hours of incubation period ( late log phase ). the eh of bhi broth was adjusted by adding an oxidizing agent , 0 . 20 m - potassium ferricyanide ( fisher scientific ), or reducing agents , 0 . 06 m - cysteine hcl , 0 . 13 m - dithiothreitol ( dtt ; sigma chemical co ., st . louis , mo . ), or 0 . 10 m - titanium ( iii ) citrate ( tc ; fisher scientific , fair lawn , n . j .) through a programmable , microprocessor controlled pipette ( hamilton bonadaz ag , switzerland ). eh was measured with a platinum combination electrode with ag / agcl as reference ( corning glass works , corning , n . y .). the electrode was calibrated by adjusting the eh value to + 86 ± 10 mv in a 0 . 05 m - potassium phosphate monobasic sodium hydroxide buffer ( ph 7 . 0 ) saturated with quinhydrone ( eastman kodak co ., rochester , n . y .) at 25 ° c . all measurements were performed in an anaerobic glove box ( forma scientific , inc ., marietta , ohio ). eh values were calculated according to the following formula ( hentges and maier , 1972 ; segel , 1976 ): eh 7 = the redox potential in relation to the hydrogen electrode at ph 7 . 0 ; e = the redox potential value measured by platinum combination electrode with ag / agcl as reference ; to determine the influence of eh on growth and leukotoxicity , f . necrophorum strain 25 was grown in medium with eh ranging from + 375 to - 352 mv . after 6 - 8 hours of incubation , samples were taken for leukotoxin assay . f . necrophorum strain 25 was grown in anaerobic bhi broth containing different concentrations of iron . all glassware and stoppers used in this experiment were soaked in 1 m hcl overnight and then rinsed thoroughly with deionized distilled water . chelex 100 resin ( 50 - 100 mesh ; bio - rad laboratories , rochville center , n . y .) was used to deplete iron from the medium by the method of murphy et al . ( 1978 ) with the following exceptions ; ( i ) a 5 - g protein of resin was added to 100 ml of bhi broth and stirred for 1 hour ; ( ii ) resin was removed by filtration , and the procedure was repeated twice , the resulting iron - limited medium (& lt ; 0 . 2 μm ) was supplemented with ca 2 + and mg 2 + ( 6 . 0 mg of cacl 2 · 2h 2 o and 2 . 2 mg of mgso 4 per 100 ml of medium ). ferric chloride solution ( fecl 3 · 6h 2 o ) was added to the iron - limited medium to obtain concentrations of 4 . 2 , 42 . 1 and 361 . 4 μm of fe 3 + . the medium was then boiled , added with cysteine hcl , and anaerobically sterilized . iron content in the medium was determined in an atomic absorption spectrophotometer . each experiment was replicated four times . data was analyzed by the general linear models procedure of sas ( 1987 ). duncan &# 39 ; s multiple range test was used for group comparison . the significance level for all analysis was p & lt ; 0 . 05 unless otherwise stated . the production of toxin by both biotypes increased with increasing bacterial growth , peaked at late - log and early - stationary phases , and then declined precipitously ( fig1 a and 1b ). the leukotoxicity almost completely disappeared after 16 hours incubation . culture supernates from biotype a had higher leukotoxin titer at all growth phases , although both biotypes had a similar cell density . the average leukotoxin titers for 11 biotype a strains and 16 biotype b strains were 882 and 56 , respectively . the ranges of leukotoxin titer for biotype a and biotype b were 160 - 1172 and 0 - 162 , respectively . four out of 16 biotype b strains did not produce detectable levels of leukotoxin . among the cultural media tested , bhi and liver infusion broths supported highest bacterial growth ( fig2 a ) the specific growth rates were 0 . 69 h - 1 and 0 . 70 h - 1 , with doubling times of 60 minutes and 59 minutes , in bhi and liver infusion broths , respectively . however , leukotoxin activity was higher ( p & lt ; 0 . 05 ) in culture grown in bhi than in liver infusion broth . f . necrophorum grew slowly ( specific growth rate = 0 . 55 h - 1 and doubling time = 76 minutes ) in eugon broth and produced minimal toxin . ph of medium from 6 . 7 to 8 . 2 had no (& lt ; 0 . 05 ) effect on the growth rate , but leukotoxicity was lower ( p & lt ; 0 . 1 ) in the culture with ph 8 . 2 than in that with ph 6 . 7 ( fig2 ). the maximum leukotoxin activity was obtained in the culture incubated 39 ° c ., which was the optimum temperature for growth ( fig2 ). no growth was observed at 43 ° c . both growth rate and leukotoxin titer were low in oxidized bhi broth ( table 1 ). however , the addition of potassium ferricyanide to increase medium eh from + 170 to + 375 mv had no ( p & lt ; 0 . 05 ) additional effect on the growth rate and leukotoxicity . bacterial growth rate in anaerobic medium without any reducing agent was low ( 0 . 40 h - 1 ). the addition of reducing agents , increased ( p & lt ; 0 . 05 ) the growth rate except , tc at low ( 0 . 32 mm ) and dtt at high ( 3 . 8 mm ) concentrations . the optimal eh for f . necrophorum growth appeared to be in the range of - 230 to - 280 mv , which required 1 . 4 μm of cysteine hcl , 3 . 2 μm of dtt , or 1 . 0 μm tc in the medium . these are within the range of concentrations normally used for anaerobic media ( costilow , 1981 ). the addition of cysteine hcl to the medium gave maximal leukotoxin titer ( 974 - 1 , 413 ), which was reflective of the high growth rate ( 0 . 50 - 0 . 60 h 1 ). however , leukotoxicity in the cultures containing tc and dtt did not correspond to the growth rate . dtt ( at 7 . 78 mm ) and tc supported bacterial growth but caused low leukotoxin titer . in tc - reduced medium , leukotoxin titer decreased as the concentration of tc increased . iron - exchange resin treatment of bhi broth decreased medium iron concentration from 6 . 1 μm to & lt ; 0 . 2 μm . in preliminary studies , resin - treated medium did not support f . necrophorum growth unless ca 2 + and mg 2 + were added to it . f . necrophorum exhibited slower ( p & lt ; o . 05 ) growth rate in the iron - limited medium than in media supplemented with iron ( fig3 ). however , growth rate was not influenced by iron concentration in the supplemented media . leukotoxin titers were similar in media containing iron at or below 42 . 1 μm . although medium with 361 . 4 μm of iron supported bacterial growth , no leukotoxicity was detected in the culture supernatant . table 1__________________________________________________________________________effect of culture medium eh on growthand leukotoxicity of f . necrophorum specificmedium and oxidizing or concentration eh growth rate leukotoxinreducing agent added ( mm ) ( mv ) ( h . sup .- 1 ) titer__________________________________________________________________________aerobic medium . sup . anone 0 + 170 . 33 86potassium ferricyanide . 77 + 375 . 28 60anaerobic medium . sup . bnone 0 . - 103 . 40 971cysteine hcl . 14 - 179 . 50 . sup . c 974 . 57 - 238 . 54 . sup . c 1286 . sup . c 1 . 42 . sup . d - 242 . 63 . sup . c 1348 . sup . c 3 . 41 - 249 . 60 . sup . c 1413 . sup . cdithiothreitol . 32 - 155 . 48 . sup . c 896 1 . 30 - 230 . 57 . sup . c 1040 3 . 24 . sup . d - 276 . 57 . sup . c 1063 7 . 78 - 325 . 53 . sup . c 327 . sup . ctitanium ( iii ) citrate . 25 - 106 . 45 226 . sup . c . 50 . sup . d - 147 . 50 . sup . c 76 . sup . c . 99 . sup . d - 217 . 51 . sup . c 76 . sup . c 1 . 96 . sup . d - 262 . 59 . sup . c 40 . sup . c 2 . 91 - 310 . 52 . sup . c 10 . sup . c 3 . 85 - 352 . 43 4 . sup . c__________________________________________________________________________ . sup . a bhi broth was not boiled to remove dissolved oxygen . . sup . b bhi broth was boiled until reeazurin turned from purple to colorless . . sup . c growth rate or leukotoxicity in anaerobic medium with reducing agent was different ( p & lt ; 0 . 05 ) from that without agent added . . sup . d concentration normally used to prepare anaerobic medium . previous studies on leukotoxin of f . necrophorum have used culture after 18 hours ( roberts , 1970 ; coyle - dennis and lauerman , 1978 ; emery et al ., 1984 ; emery et al ., 1986 ), 3 - 4 days ( scanlan et al ., 1982 ), or 7 days ( fales et al ., 1977 ) of incubation . emery et al . ( 1984 ) have indicated that leukotoxin production was critically affected by cell concentration and period of incubation . leukotoxin titer was above 1 , 000 in 18 - hour culture , with an approximate bacterial concentration of 10 9 cells / ml , but 3 - d culture superhate had much lower leukotoxicity . in the present example , maximal toxin production occurred at late - log and early stationary phases and was followed by a precipitous decline in leukotoxicity in both a and b biotypes . as indicated , the rapid decrease in leukotoxicity after stationary - phase may have been due to degradation of toxin possibly by proteolytic enzyme ( s ) produced by the bacteria . f . necrophorum produces proteolytic enzymes ( wallace and brammall , 1985 ). because leukotoxin is a protein ( garcia et al ., 1975a ; coyle - dennis and lauerman , 1978 ) inactivation by proteolytic enzymes appears to be the logical reason for precipitous decline in leukotoxicity . scanlan et al . ( 1986 ) reported that f . necrophorum biotype a produced more leukotoxin that biotypes ab and b . the percentages of macrophage death by culture filtrate from biotypes a , ab and b were 93 %, 77 % and 21 % respectively . in the present study with 27 strains , the average of leukotoxin titer was approximately 18 times higher for biotype a than that for biotype b . the difference in toxin production by biotypes may account for the fact that biotype a is more virulent and more frequently encountered in hepatic abscesses than biotype b ( scanlan and hathcock , 1983 ). although samples for toxin assay were obtained at the same growth stage ( late - log phase ), a considerable strain - to - strain variation in toxin production was observed . toxin titers were in excess of 1 , 000 in some strains whereas no toxicity was detected in others . the nonleukotoxin - producing strains were hemolytic and formed sediment in the liquid medium , and thus did not meet the criteria for nonpathogenic biotype c ( fievez , 1963 ). emery et al . ( 1984 ) reported that leukotoxin production was higher in eugon or modified eugon broth than in nutrient or thioglycollate broth . however , in the present example , eugon broth was the least supportive of the three media tested for growth and leukotoxin production while bhi was the most supportive . the reason for the influence of medium on toxin production was unclear . various culture media have been used to study leukotoxin production by f . necrophorum . these included m - 1 continuous dialysis sac culture ( fales et al ., 1977 ; scanlan et al ., 1982 ), eagles minimal essential medium ( fales et al ., 1977 ), modified thioglycollate broth ( coyle - dennis and lauerman , 1979 ), bhi agar ( scanlan et al ., 1982 ) and modified heart infusion broth ( kanoe et al ., 1985 ), medium with ph 8 . 2 supported f . necrophorum growth but not leukotoxin production . the stability of leukotoxin to extreme ph ( 4 . 0 - 9 . 0 ) has been reported ( emery et al ., 1984 ; scanlan et al ., 1986 ). therefore , high ph may affect production rather than activity of the leukotoxin . because liver is a highly vascularized organ , with blood eh around + 126 to + 422 ( meynell , 1963 ), it is of interest to know the influence of culture medium eh on growth and leukotoxin production by f . necrophorum . a number of investigators ( hentges and maier , 1972 ) have suggested that the growth of anaerobic bacteria is primarily effected by eh of the medium . in contrast , it is postulated that the efficacy of reducing agents in promoting the growth of anaerobic organisms is due entirely to their oxygen - removing properties ( o &# 39 ; brien and morris , 1971 ); both may be important ( hentges and maier , 1972 ). in this example , oxygen was excluded by boiling and with the use of oxygen - free co 2 prior to adding reducing agent . therefore , the higher growth rate in low - eh medium , indicated that eh did effect growth . in order to grow and set up infection in liver . f . necrophorum has to overcome the aerobic environment . aerobic or facultative bacteria , such as actinomyces pyogenes and streptococcus spp , have been isolated frequently with f . necrophorum in liver abscesses ( kanoe et al ., 1976 ; berg and scanlan , 1982 ; lechtenberg et al ., 1988 ). it is postulated that a synergistic interaction exists between f . necrophorum and facultative bacteria in the liver . the facultative organisms may utilize oxygen and lower eh in host tissue to enable f . necrophorum growth ( beveridge , 1934 ; roberts , 1970 ; takeuchi et al ., 1983 ; brook and walker , 1984 ). however , f . necrophorum is often isolated in pure culture in liver abscesses . also , liver abscesses in mice and cattle have been experimentally induced by the injection of f . necrophorum alone ( takeuchi et al ., 1984 ; itabisashi et al ., 1987 ); lechtenberg and nagaraja , 1991 ). it is known that f . necrophorum has a potent endotoxic lipopolysaccharide ( hofstad and kristoffersen , 1971 ; garcia et al ., 1975b ; warner et al ., 1975 ; berg and scanlan , 1982 ). this endotoxin and other components such as hemagglutinin of f . necrophorum cause platelet aggregation and may induce focal intravascular coagulation ( forrester et al ., 1985 ; kanoe and yamanka , 1989 ). this may result in an anaerobic microenvironment in which f . necrophorum will grow . platelet aggregation is induced only by biotype a ( forrester et al ., 1985 ), which explains why biotype a is frequently isolated in pure culture ( berg and scanlan , 1982 ); lechtenberg et al ., 1988 ). a high concentration of tc ( 3 . 0 mm ) had a detrimental effect on the growth of f . necrophorum . the inhibitory effect of tc on ruminal bacteria has been reported ( wachenheim and hespell , 1984 ). the decrease in leukotoxicity was related to the strength and concentration of the reducing agent . tc was the strongest reducing agent among the three and inactivated the toxin greatly even at a low concentration ( 0 . 25 mm ). presumably , reducing agents structurally alter the toxin , a protein substance , by reducing disulfide bonds . as with many other pathogenic bacteria ( bjorn et al ., 1979 ; filed et al ., 1986 ; kadurugamuwa et al ., 1987 ) f . necrophorum requires iron as a growth factor . the availability of iron to microorganism is limited because of the competition from host iron - binding proteins such as transfertin and lactoferrin ( finkelstein et al ., 1983 ). to acquire the iron essential for their growth and metabolism , the microbes have to complete with the host for iron . the kemolytic activity of f . necrophorum has been , reported ( garcia et al ., 1975a ; abe et al ., 1979 ; kanoe et al ., 1984 ; emery et al ., 1985 ); hemolysins break down erythrocytes and cause iron release . therefore , the hemolytic activity of f . necrophorum may possibly aid in acquiring iron from the host . also , it has been demonstrated that some pathogenic bacteria produce iron chelators ( siderophores ) to acquired iron ( finkelstein et al ., 1983 ; field et al ., 1986 ). whether or not f . necrophorum produces iron chelators is not known . the association of availability of iron with toxin production has been reported in a number of studies . gentry et al . ( 1986 ) indicated that the rate of leukotoxin production in pasteurella haemolytica was enhanced by iron - containing and iron - chelating compounds . strathdee and lo ( 1989 ) later found that the leukotoxin determinant was composed of four contiguous genes and the expression of the toxin was greatly reduced by iron limitation . in contrast , the production of diphtheria toxin in corynebacterium diphtheria ( murphy et al ., 1978 ) and of exotoxin a in pseudomonas aeruqinosa ( bjorn et al ., 1979 ; woods et al ., 1982 ) was inhibited by the presence of iron . in the present example , leukotoxicity of f . necrophorum was not affected by iron except at a high concentration ( 361 . 4 μm ). leukotoxicity was reduced but the growth of f . necrophorum was unaffected in medium containing high iron concentration . therefore , the activity rather than production of the toxin appeared to be affected by high concentration of iron . these results demonstrate that growth conditions influence leukotoxin production by f . necrophorum . however , it should be emphasized the conditions like ph and eh were not regulated or monitored through the incubation . therefore , it is likely that the initial measurements may not be reflective of the conditions existing throughout the growth period . the following example sets forth exemplary methods for the inactivation of leukotoxin supernate and inactivation of an entire culture , in order to produce vaccines for use in immunizing ruminant animals , such as cattle and sheep . inactivation of whole culture or culture superhate containing leukotoxin is preferably carried out by adding formalin ( 0 . 3 - 0 . 4 %) or b - propiolactone ( 0 . 10 - 0 . 12 %) on a vol / vol basis . inactivated whole cultures are chilled in an ice bath and refrigerated for two days . if β - propiolactone is used , any residuum thereof is hydrolyzed by heating the cultures at 39 ° c . for 4 - 6 hours . inactivation of whole cultures is tested by streaking the samples on bhi blood agar ( reduced with 0 . 05 % cysteine hydrochloridel and incubating the plates anaerobically for 24 hours . the resultant plates should exhibit no growth . the completed vaccine can be administered in a variety of ways , such as by intramuscular or parenteral injections . the following example provides exemplary methods for the production of vaccine , and the prophylactic use thereof against f . necrophorum . thirty holstein steers weighing 200 - 370 kg each were immunized with leukotoxin from f . necrophorum in a sixty - three day immuno - challenge test to determine whether serum leukotoxin neutralizing antibodies would protect against f . necrophorum . prior to the initial immunization on day zero , ultrasonographic scanning confirmed that all animals were free from liver abscesses . blood sample measurements were conducted according to the standard mtt - dye reduction neutralization assay for determination of the baseline serum leukotoxin neutralizing antibody titer . the thirty steers were randomly divided into six groups of five , and each group was injected with different inoculum preparations , as described below , through the administration of one subcutaneous injection on each of days 0 and 21 . several anaerobic broth culture tubes were prepared to hold cultures of f . necrophorum for subsequent use . the broth culture preparation process began with an existing f . necrophorum , strain 25a ( biotype a ) , culture , which was inoculated onto an anaerobic bhi blood agar , and incubated at 39 ° c . in an anaerobic glove box ( forma scientific co .) for 24 hours to obtain isolated colonies . anaerobic bhi blood agar was prepared by prereducing commercial bhi broth with 0 . 05 % by volume cysteine hydrochloride , and then dispensing 15 ml of the broth under nitrogen into each of several tubes containing respective 300mg portions of agar . additionally , a 0 . 001 % by volume portion of resazurin was included in the medium as a redox potential indicator . the tubes were closed with butyl rubber stoppers , crimped with aluminum seals , and autoclaved for 15 minutes . any tubes showing pink coloration ( a sign of oxidation ) were discarded . the autoclaved tubes were placed inside the glove box , and cattle blood was pipetted into the tube to reach a 5 % by volume blood portion ( 0 . 75 ml ). the tubes were stoppered and gently mixed by inverting the tube , and the culture therein was poured into sterile petri plates . blood agar plates were kept in the glove box , and used for culturing after at least 24 hours had passed for equilibration . single colonies of f . necrophorum from the blood agar plates were inoculated with a loop into tubes containing 10 ml of anaerobic bhi broth . this anaerobic bhi broth was prepared as before , without agar , with prereduction , and anaerobic sterilization . the inoculated broth cultures inside the respective tubes were incubated at 39 ° c . for 6 to 9 hours . during culturing , the initial ph of 7 . 5 dropped to about 6 . 8 . the preparation , dispensing , inoculation , and sample removal techniques employed were as described in holdeman et al , anaerobic laboratory manual , 4th edition , virginia polytechnical institute . the broth cultures were later used as inoculum for the introduction of f . necrophorum into larger cultures having a different medium from which leukotoxin test compositions were obtained for subsequent injection into the test animals . the test compositions included &# 34 ; inactivated cell culture ,&# 34 ; &# 34 ; crude toxin , &# 34 ; and &# 34 ; semi - purified toxin .&# 34 ; &# 34 ; inactivated cell culture &# 34 ; was prepared by treating active cell culture with formalin . anaerobic bhi medium ( vpi anaerobic culture system ) was prepared according to standard methods in a 4 liter flask containing 3 liters of medium . the flask was inoculated with 35 ml of log - phase f . necrophorum , strain 25a , culture , and incubated on a rocking platform at 39 ° c . for 6 to 8 hours ( a 660 = 0 . 6 to 0 . 75 ). the cell culture , which had a count of 2 . 4 × 10 8 cfu / ml , was inactivated through the addition of formalin to achieve a 0 . 3 % ( v / v ) formalin concentration . &# 34 ; crude toxin &# 34 ; was produced from filter - sterilized culture supernatant . cell culture was prepared in the same manner as for inactivated cell culture . supernatant was decanted after centrifugation of active culture at 13 , 500 g for 15 minutes ( at 4 ° c . to protect the leukotoxin ), and 1 ml was retained for a test of the leukotoxin activity . the decanted supernatant was inactivated through the addition of formalin until the formalin concentration reached 0 . 3 % ( v / v ). the inactivated supernatant was sterilized by filtration through a 0 . 45 μm membrane at 4 ° c . &# 34 ; semi - purified toxin &# 34 ; was prepared as a gel filtrate of concentrated crude toxin . crude toxin was prepared as before , except in a 6 liter culture volume ( two flasks ). the crude toxin was subsequently subjected to concentration and filtration procedures to obtain semi - purified toxin as described in the paragraphs below . in the concentration step , a 10 k hollow fiber filter was installed in a hollow fiber concentration / desalting unit ( amicon dc10 ) according to the manufacturer &# 39 ; s instructions . the filters were washed and conditioned in a series of steps : washing with 4 liters of distilled water ; circulating 2 liters of double distilled water for at least one hour ; and circulating 2 liters of pbs ( ph 7 . 4 ) for at least 2 hours . after conditioning of the filters , the unit concentrated the 6 liters of culture supernatant filtrate to 100 ml at 4 ° c . the concentrated leukotoxin was dispensed in 3 ml aliquots and stored at - 70 ° c . two aliquots were retained for determination of leukotoxin titer and protein concentration . the gel filtration step employed sephacryl s300 gel from pharmacia , which is suitable for molecular separation sizes ranging from 1 × 10 4 to 1 . 5 × 10 6 . the filtration step used an elution buffer mixed with filter - sterilized double distilled water , phosphate buffer saline solution (&# 34 ; pbs &# 34 ;) at ph 7 . 4 , 10 μm cacl 2 , and 10 μm mgcl 2 . the entire filtration procedure was performed at 4 ° c . in order to maintain the leukotoxin activity . the gel was packed in an xk 26 / 70 column according to the manufacturer &# 39 ; s instructions . the lower end of the column was connected to a uv monitor and fraction collector . the column was pre - equilibrated with 350 ml of buffer , and 0 . 002 % ( w / v ) blue dextran was added for determination of the column void volume . the concentrated leukotoxin was mixed with a 50 % glycerol solution ( aq .) in a 9 : 1 ratio ( v / v ) immediately prior to filtration . the 9 part volume of concentrated leukotoxin was selected to contain approximately 200 mg protein . the column was loaded with the mixture , which was filtered at a rate of 30 ml per hour . five ml fractions were collected after the protein began to elute from the column . group 1 was injected , in each instance , with 20 ml of vaccine formed from inactivated cell culture that was mixed with ribi adjuvant -- an oil emulsion adjuvant prepared by ribi immunochem of hamilton , mont . the ribi adjuvant included 10 % drakeol 6vr light mineral oil ( from penreco of butler , pa . ), 12 % ( w / v ) lecithin ( centrolex p from central soya of fort wayne indiana ), and 2 . 0 mg / ml synthetic trehalose dicorynemycolate ( s - tdcm from ribi immunochem ). the inactivated culture was diluted with sterile saline containing 0 . 4 % ( v / v ) tween 80 to a concentration equating to 2 . 4 × 10 8 cfu / ml , mixed with 10 % ( v / v ) of ribi adjuvant , and emulsified at 4 ° c . each steer in group 2 was injected with 20 ml of crude , toxin mixed with ribi adjuvant . the crude toxin included 0 . 6 mg protein / ml or 15 , 640 leukotoxin units per ml before formalin treatment , which was mixed with 10 % ( v / v ) of ribi adjuvant per injection . each steer in group 3 was injected with 4 . 6 ml of semi - purified toxin mixed with ribi adjuvant . the toxin preparation contained 10 mg protein ( or 1 , 022 , 994 leukotoxin units ) before formalin treatment , which was mixed with 10 % ( v / v ) of ribi adjuvant per injection . the steers of group 4 were each injected with 4 . 6 ml of semi - purified toxin mixed with stimulon - 21 adjuvant ( qs - 21 from cambridge biotech corporation of worcester , mass .). the toxin preparation contained 10 mg protein ( or 1 , 022 , 994 leukotoxin units ) before formalin treatment , and the same was mixed with 100 μg of stimulon - 21 adjuvant per injection . the steers of group 5 were used as tylosin antibiotic ( tylan from elanco animal health , greenfield , ind .) control animals . these were each injected with 10 ml of a solution containing pbs mixed with 10 % ( v / v ) ribi adjuvant . subsequently , the steers were fed 100 mg of tylosin antibiotic per day mixed with 0 . 5 kg of ground corn . the group 6 steers were pbs controls that were created by mixing pbs with 10 % ( v / v ) ribi adjuvant and injecting 10 ml of this solution into the control animals . adjuvants , such as ribi and stimulon , work to promote antibody production in a nonspecific manner and , consequently , to enhance the general level of induced antibody production . as a measurement of this response , serum leukotoxin neutralizing antibody titers were monitored in the test animals throughout the study . after inoculation with leukotoxin on day zero , blood samples were collected every seven days until 42 days had passed , and the samples were tested to monitor the serum leukotoxin neutralizing antibody titer . table 2 sets forth the results , which demonstrate that a maximum titer of 59 , 278 occurred for the crude supernatant plus ribi group during week three . table 2__________________________________________________________________________serum leukotoxin neutralizing antibody titertest grouping * ( 2 ) ( 3 ) ( 4 ) ( 1 ) crude supernatant semi - purified semi - purified toxin ( 5 ) ( 6 ) week whole cell culture plus ribi toxin plus ribi plus stimulon tylosin contr__________________________________________________________________________0 19 36 10 26 26 261 97 1 , 572 185 6 , 063 24 1252 765 5 , 378 2 , 122 13 , 767 64 1103 2 , 893 59 , 278 13 , 905 6 , 374 187 1164 4 . 675 17 , 327 5 , 271 11 , 849 138 675 3 , 533 5 , 541 3 , 134 14 , 320 171 726 2 , 100 9 , 136 4 , 447 14 , 618 48 176__________________________________________________________________________ * vaccines were given on days 0 and 21 . on day 42 , through the use of an ultrasound - guided percutaneous catheterization procedure , the immune response of each steer was challenged by injecting inoculum taken from an active , seven hour culture of f . necrophorum , strain 25a . various comparative methods were thereafter employed to monitor the impact of f . necrophorum infection upon the challenged animals . the liver of each steer was ultrasonographically scanned on days 42 , 49 , and 63 . after the last scan , the steers were euthanized and necropsied to examine the respective livers for abscesses . the results are presented in table 3 below . the group 2 results indicate that crude toxin mixed with ribi constituted the most effective prophylactic with regard to the prevention of liver abscesses , i . e ., no incidence of liver abscesses as confirmed by necropsy versus 100 % in the pbs control . table 3__________________________________________________________________________induced liver abscessresults comparison number of liver abscessesgroup animals mortality ultrasonography necropsy incidence__________________________________________________________________________ ( 1 ) whole cell 5 1 * 2 / 4 3 / 5 60culture plus ribi ( 2 ) culture super - 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