bacterial strains , growth conditions , and the peptide : b. abortus biotype 1 isolate from korean cattle was used for the 
 construction of a vaccine by means of gi24 . b. abortus strain rb51 served as the comparative vaccine ( versus the vaccine candidate ) . b. 
 abortus strain 544 ( atcc 23448)smooth , virulent bacteria of the b. abortus biovar 1 strain  served as the virulent challenge strain 
 ( table 1table 1.bacterial strains and plasmids used in this studystrain / plasmiddescriptionsource or referencestrainsb . abortusbiotype 1isolate from korean cattlelab stockstrain rb51commercial vaccine strainlab stockstrain 544atcc23448 , a smooth virulent b. abortus biovar 1 strain ) . these strains were grown in brucella broth and on brucella agar ( becton dickinson , sparks , md , u.s.a . ) 
 at 37c . the gi24 ( grfrrlrkktrkrlkkigkvlkwi - nh2 ) peptide was chemically synthesized by peptron ( daejeon , south korea ) . the b. 
 abortus biotype 1 isolate was kindly supplied by the national veterinary research and quarantine service ( nvrqs ) , anyang , korea , after 
 identification of the phenotypic characteristics of the b.abortus biotype 1 isolate by typical biochemical tests and 
 brucella biotyping using the co2 requirement for primary isolation , h2s production , urease activity , growth in the 
 presence of dyes , agglutination with monospecific sera , phage typing and oxidative metabolic rates . construction of a b. abortus vaccine candidate : a single colony ( round with smooth margins and approximately 2 mm in diameter ) 
 of b. abortus biotype 1 isolate was individually inoculated into 200 ml of brucella broth , and the cultures were incubated at 
 37c with slow agitation to obtain optical density ( od ) of 0.3 at 600 nm . gi24 was added into the culture broth at 40 g / ml and 
 was incubated at 37c to lyse the isolate . after 24 hr , lysis induction was determined by counting the number of viable cells after incubation on brucella agar 
 for 72 hr at 37c . after the lysis process , the cell lysates were harvested by centrifugation at 4,000  g for 10 min . finally , the harvested 
 cells were washed three times with sterile phosphate - buffered saline ( pbs ) , then resuspended in pbs at the concentration of approximately 3  10 
 cells / ml and stored at 20c . preparation of a b. abortus strain rb51 formulation : strain rb51 was cultured in brucella broth for 48 hr at 37c . the bacteria 
 were washed with sterile pbs three times and resuspended in sterile pbs at approximately 3  10 colony - forming units ( cfus)/ml . transmission electron microscopy ( tem ) : the lysate samples for tem to examine the intracellular alterations in the b. abortus 
 cells before and after treatment with peptide gi24 were prepared in the same manner as the b. abortus lysates . immunization and sample collection : balb / c mice were subdivided into four groups , each group containing 10 mice . all the mice were inoculated at 
 6 weeks of age [ 0 week postinoculation ( wpi ) ] . ten mice of group a were inoculated ip with sterile pbs , as the control group was . mice of groups b and c were also 
 vaccinated ip with approximately 3  10 cfus of b. abortus strain rb51   and approximately 
 3  10 cells of the brucella vaccine candidate . group d was orally immunized with approximately 3  10 cells of the 
 vaccine candidate . blood and vaginal washing samples were collected 0 , 2 , 4 and 6 wpi for evaluation of the immune response according to the method described in 
 another study . the animal experiments described in this study were conducted with approval ( cbu 2015 - 052 ) of the 
 chonbuk national university animal ethics committee in accordance with the guidelines of the korean council on animal care . immune responses evaluated by an enzyme - linked immunosorbent assay ( elisa ) : a modified elisa was performed to assess the 
 brucella lipopolysaccharide ( lps)-specific igg and iga titers in serum and vaginal washing samples , using the b. brucella ab elisa 2.0 kit 
 ( bionote , hwaseongsi , gyeonggi - do , republic of korea ) . briefly , serum samples and vaginal washing samples were diluted 1:50 and 1:3 with pbs , respectively . the 
 plates were incubated with a horseradish peroxidase - conjugated goat anti - mouse igg or iga antibody ( southern biotechnology associates , birmingham , al , u.s.a . ) . enzymatic reactions were carried out by addition of a substrate solution containing o - phenylenediamine ( sigma - aldrich , st . louis , mo , u.s.a . ) and 
 were analyzed on an automated elisa spectrophotometer ( thermo scientific multiskan go , thermo fisher scientific oy , ratastie , vantaa , finland ) at 492 nm . results 
 of the elisa are expressed as the mean optical density ( od )  standard deviation . quantitation of cytokines from splenocytes : from each group , five mice were euthanized , and spleens were removed aseptically 4 wpi . the splenocytes were prepared according to the method described elsewhere [ 1 , 38 ] . the prepared spleen cells were seeded in 24-well tissue culture plates at 2  10/well [ 1 , 38 ] . the splenocytes were stimulated in vitro with heat - inactivated bacteria of 
 b. abortus strain 544 ( 10 cells / well ) , concanavalin a ( 0.5 g / well ) as a positive control or the medium as an 
 unstimulated control , and incubated at 37c , 5% co2 and 95% humidity [ 1 , 52 ] . culture supernatants were collected after 72 hr of restimulation and stored at 70c until use for cytokine quantification [ 1 , 52 ] . quantification of cytokines by an elisa : elisa was used to measure the concentration of interleukin ( il)-4 , il-10 , interferon - gamma ( ifn- ) , and 
 tumor necrosis factor alpha ( tnf- ) in the supernatants using the mouse cytokine elisa ready - set - go reagent kit ( ebioscience inc . , san diego , ca , u.s.a . ) [ 6 , 7 , 42 ] . challenge experiments : for challenge experiments , the challenge strain , strain 544 was prepared . briefly , the strain was grown in brucella broth 
 at 37c for 24 hr and was resuspended at approximately 10 cfus / ml . all mice were challenged ip 6 wpi with 100 l of 
 the challenge strain . two weeks after the challenge , the spleen weights of all mice were measured , and each spleen was diluted 1:100 using brucella broth . a total 
 of 100 l of the diluted media was spread on blood agar to count the number of viable strain 544 cells from the spleens at 2 weeks after the 
 challenge . if no colony was detected on the blood agar , then the number of viable cells of challenge strains from the spleen of the mice corresponded to 
 < 10 cfus . briefly , blended spleen samples were plated on brucella agar with or without rifampicin ( 50 g / ml ) , 
 because strain rb51 is resistant to rifampicin . the vaccine and challenge strains were confirmed by pcr using a 
 b. abortus - specific primer ( 5-gac gaa cgg aat ttt tcc aat ccc ) , rb51/2308 primer ( 5-ccc cgg aag ata tgc ttc gat cc ) and 
 is711-specific primer ( 5-tgc cga tca ctt aag ggc ctt cat ) with enhanced brucella amos pcr primers [ 10 , 11 ] . statistical analysis : to evaluate the differences among various vaccinated groups , absorbance data from elisas were subjected to analysis of 
 variance with post hoc tukey s test for pairwise comparison in the spss software , version 16.0 ( spss inc . , chicago , il , u.s.a . ) . the 
 kruskal - wallis test followed by dunn s procedure for multiple comparisons between groups was carried out to compare the log - transformed cfu values in organs 
 obtained from each vaccinated group of mice with the values in the respective control group in the bacterial challenge experiments . the bacterial cells were incubated with 40 g / ml of gi24 for 30 hr at 37c . , in the untreated b. abortus cells , full intracellular contents and an intact cell membrane were observed ( fig . 1b ) , the bacteria treated with gi24 showed clear cytoplasmic 
 spaces , and a disrupted membrane of b. abortus ( with visible pores ) was observed . the bacterial cells were incubated with 40 g / ml of gi24 for 30 hr at 37c . the humoral immune response in immunized mice : serum igg titers against b. abortus lps in groups c and d were 0.07  0.009 and 
 0.08  0.025 before immunization and increased gradually to 1.78  0.484 and 1.66  0.407 at 6 wpi ( fig . 2.serum igg ( g / ml ) and vaginal iga ( g / ml ) titers against brucella 
 abortus 1119 - 3 lps in mice intraperitoneally or orally immunized with each vaccine candidate . group a mice were immunized with sterile pbs ; group 
 b mice were intraperitoneally immunized with 3  10 cfus of b. abortus strain rb51 ; group c mice were intraperitoneally 
 immunized with 3  10 cells of the b. abortus lysates ; group d mice were orally immunized with 3  10 cells of the 
 b. abortus lysates . data shown are the means of all mice in each group , and error bars show the standard deviations ( sd ) . asterisks 
 indicate a significant difference between the values of the groups immunized with the vaccine candidate ( * p0.05 ) and those of the control 
 group . ; p<0.01 ) . in addition , the vaginal iga titers against the b. abortus lps in groups b , c and d were 0.13  0.037 , 0.14 
  0.033 and 0.11  0.025 , respectively , before immunization and 0.2  0.061 , 0.7  0.239 and 0.36  0.185 , respectively , 2 wpi . four wpi , the iga titers of groups 
 b , c and d were 0.44  0.093 , 0.66  0.195 and 0.45  0.108 , respectively ( p<0.01 ) . the iga titers of groups b , c and d were 0.23  0.036 , 
 0.87  0.089 and 0.36  0.103 , respectively , 6 wpi ( fig . 2 ; p<0.01 ) . serum igg ( g / ml ) and vaginal iga ( g / ml ) titers against brucella 
 abortus 1119 - 3 lps in mice intraperitoneally or orally immunized with each vaccine candidate . group a mice were immunized with sterile pbs ; group 
 b mice were intraperitoneally immunized with 3  10 cfus of b. abortus strain rb51 ; group c mice were intraperitoneally 
 immunized with 3  10 cells of the b. abortus lysates ; group d mice were orally immunized with 3  10 cells of the 
 b. abortus lysates . data shown are the means of all mice in each group , and error bars show the standard deviations ( sd ) . asterisks 
 indicate a significant difference between the values of the groups immunized with the vaccine candidate ( * p0.05 ) and those of the control 
 group . cytokine analysis : the mean concentrations of il-4 in response to heat - inactivated b. abortus cells from splenocytes of mice 
 from groups a , b , c and d were 25  8.477 pg / ml , 112.1  21.129 pg / ml , 168.7  36.53 
 pg / ml and 199  63.116 pg / ml , respectively ( p<0.05 ) ( fig . 3fig . 3.il-4 ( pg / ml ) , il-10 ( pg / ml ) , tnf - a ( pg / ml ) and ifn- 
 ( ng / ml ) concentrations in the supernatants of the splenocytes stimulated with heat - inactivated brucella 
 abortus strain 544 at 4 wpi . group a mice were immunized with sterile pbs ; group b mice were intraperitoneally immunized with 3  10 
 cfus of b. abortus strain rb51 ; group c mice were intraperitoneally immunized with 3  10 cells of the b. 
 abortus lysates ; group d mice were orally immunized with 3  10 cells of the b. abortus lysates . data are presented 
 as the mean of all mice in each group ; error bars show sd . asterisks indicate a significant difference between the values of mouse groups b , c and d 
 ( * p<0.05 ) and those of the control group . ) . the il-10 concentrations of the mice from groups a , b , c and d were 17.4  10.644 pg / ml , 72.1  21.829 
 pg / ml , 101.5  39.049 pg / ml and 92.5  48.855 pg / ml , 
 respectively ( p<0.05 ) . the tnf- concentration of mouse groups a , b , c and d were 162.5  18.208 pg / ml , 
 375.8  76.996 pg / ml , 276.3  61.266 pg / ml and 316  87.429 
 pg / ml , respectively ( p<0.05 ) . in addition , the levels of ifn- in groups a , b , c and d were 1  0.385 
 ng / ml , 3.7  1.29 ng / ml , 2.4  0.274 ng / ml and 1.6  0.091 
 ng / ml , respectively ( p<0.01 ) . il-4 ( pg / ml ) , il-10 ( pg / ml ) , tnf - a ( pg / ml ) and ifn- 
 ( ng / ml ) concentrations in the supernatants of the splenocytes stimulated with heat - inactivated brucella 
 abortus strain 544 at 4 wpi . group a mice were immunized with sterile pbs ; group b mice were intraperitoneally immunized with 3  10 
 cfus of b. abortus strain rb51 ; group c mice were intraperitoneally immunized with 3  10 cells of the b. 
 abortus lysates ; group d mice were orally immunized with 3  10 cells of the b. abortus lysates . data are presented 
 as the mean of all mice in each group ; error bars show sd . asterisks indicate a significant difference between the values of mouse groups b , c and d 
 ( * p<0.05 ) and those of the control group . protection of mice against a virulent challenge : all mice were challenged ip 6 wpi with approximately 4  10 cfus of the challenge 
 strain . among the five mice of group a , the challenge strain was isolated from all mice , and the number of isolates was 115,296  15,446 cfus / spleen . the 
 challenge strain was isolated from all mice of group b , and the number was 19,243  9,229 cfus / spleen . in contrast , in group c , the challenge strain was isolated 
 from three out of five mice , and the number was only 4,267  2,512 cfus / spleen . among four of five mice in group d , the wild - type b. abortus 
 strain was isolated , and the number was 13,118  1,005 cfus / spleen ( fig . 4.protection against b. abortus strain 544 in mice immunized with b. abortus strain rb51 or b. abortus 
 ghost cells . group a mice were immunized with sterile pbs ; group b mice were intraperitoneally immunized with 3  10 cfus of b. 
 abortus strain rb51 ; group c mice were intraperitoneally immunized with 3  10 cells of the b. abortus lysates ; and 
 group d mice were orally immunized with 3  10 cells of the b. abortus lysates . six weeks postinoculation ( wpi ) , the mice were 
 challenged with b. abortus strain 544 . two weeks later , the number of viable brucella cells recovered from the spleen was 
 determined . data represent the mean of all mice in each group ; error bars show sd . asterisks indicate a significant difference between the values of mouse 
 groups b , c and d ( * p<0.05 ) and those of the control group . ) . protection against b. abortus strain 544 in mice immunized with b. abortus strain rb51 or b. abortus 
 ghost cells . group a mice were immunized with sterile pbs ; group b mice were intraperitoneally immunized with 3  10 cfus of b. 
 abortus strain rb51 ; group c mice were intraperitoneally immunized with 3  10 cells of the b. abortus lysates ; and 
 group d mice were orally immunized with 3  10 cells of the b. abortus lysates . six weeks postinoculation ( wpi ) , the mice were 
 challenged with b. abortus strain 544 . two weeks later , the number of viable brucella cells recovered from the spleen was 
 determined . data represent the mean of all mice in each group ; error bars show sd . asterisks indicate a significant difference between the values of mouse 
 groups b , c and d ( * p<0.05 ) and those of the control group . a novel method for production of an inactivated b. abortus vaccine candidate is based on b. abortus lysis using one of the amps 
 leading to formation of empty cell envelops , termed  brucella lysate vaccine  . the peptides bearing a net positive charge get attached to the 
 negatively charged phospholipids in the cell membranes of gram - negative and gram - positive bacteria [ 23 , 48 ] , followed by insertion of the peptide into to the bacterial cell membrane , 
 resulting in formation of a transmembrane  pore  [ 20 , 37 ] . as soon as pores form , 
 they disrupt and destabilize the bacterial cell membrane , and then all cytoplasmic content comes out leading to bacterial lysis [ 20 , 37 ] , with intact natural outer membrane lps structures that have highly immunogenic properties . vaccination via the mucosal route is a feasible method to induce acquired immune responses against infectious agents in humans and animals . in most animals , live 
 attenuated brucella strains were used as mucosal vaccine candidates in several studies [ 14 , 21 , 36 ] . however , the inherent safety risks , such as bacterial replication , may prevent 
 the use of live attenuated brucella strains as vaccines . other studies showed that salmonella gallinarum cell lysates can 
 generate protective antibody - mediated as well as cell - mediated immunity ( cmi ) when used as a vaccine via various routes in chickens [ 13 , 22 ] . bacterial lysates represent a relatively new concept for improvement of the vaccine technology , 
 but this novel approach has been seldom tested on brucella . as reported in this study , we constructed a brucella vaccine 
 candidate using gi24 , one of amps . in this study , we tested whether the brucella vaccine candidate can be administered orally as well as ip to 
 protect mice from a challenge infection with wild - type b. abortus . we investigated the efficacy of the brucella vaccine 
 candidate , constructed using gi24 , for protection against brucellosis caused by strain rb51 in murine models . we first examined the effect of the 
 brucella vaccine candidate ( constructed using gi24 ) on induction of cmi and protection . in line with our initial hypothesis , ip or oral 
 immunization with the brucella vaccine candidate significantly increased antibody titers and cmi responses in comparison with the unimmunized 
 group . in the present study , we evaluated humoral immunity as well as cmi induced by immunization with the brucella vaccine candidate and its 
 protective efficacy against challenge infection with b. abortus strain 544 . the route of immunization alters the course of the immune response 
 being induced by the vaccine candidate . therefore , we evaluated two routes of immunization , ip and oral , to find the optimal immunization route required for an 
 effective immune response . approximately 3  10 and 3  10 cells of the brucella vaccine candidate were used as the dose 
 for immunization via ip and oral routes , respectively . a vaccine inducing protective immune responses , such as secretory iga , at the mucosal surface would be an 
 ideal vaccine , because it would prevent the entry of ( and colonization by ) pathogens and consequently would prevent the disease . in the present study , immune responses , such as serum igg and mucosal iga , the serum igg and vaginal iga titers in the vaccine candidate - inoculated groups irrespective of the immunization route were significantly increased as 
 compared to the unimmunized group . furthermore , induction of cytokines in splenocytes collected from all mice  immunized with the vaccine candidate or strain rb51 
 and restimulated in vitro with heat - inactivated bacteria of b. abortus strain 544showed powerful th2 type immunity ( il-4 levels 
 represent the th2 type of immunity ) . generally , induction of mucosal iga is strongly dependent on a type th2 immune response . major cytokines that enhance iga 
 responses are il-5 , il-6 and il-10 [ 33 , 44 ] . in this study , il-10 levels were 
 significantly higher in all the mice immunized the brucella vaccine candidate than in control mice . these results show that the production of 
 cytokines that are associated with enhancement of an igg and iga response was also significantly increased by the brucella vaccine candidate . it is well known that brucella is a facultative intracellular pathogen . for clearance of an intracellular pathogen , therefore , in this study , we evaluated cellular immune responses using elisa kits 
 for cytokines . the results revealed strong secretion of tnf- and ifn- into the culture supernatant when splenocytes were restimulated in vitro , 
 giving an indication of the th1 type of immune response ; this result is in agreement with the previously shown protective response . for efficient macrophage 
 functioning , such as killing and clearance of an intracellular pathogen , ifn- is an essential effector cytokine [ 32 , 43 ] . in the present study , a strong th1 response ( tnf- and ifn- ) was induced by ip immunization with strain rb51 or by ip or 
 oral vaccination with the brucella vaccine candidate . these results showed that immunization with the brucella vaccine candidate 
 irrespective of the immunization route effectively induces the cytokines that are related to cmi as well as humoral immunity . in mouse brucellosis models , the vaccine - induced protective immune responses are analyzed by comparing a reduction in a bacterial load in the spleen , liver or 
 both between the vaccinated mice and unvaccinated animals . on the basis of that study , mice vaccinated with the 
 vaccine candidate and strain rb51 showed a decrease in the splenic counts of brucella after an ip challenge with wild - type b. 
 abortus strain 544 . nevertheless , the notable difference between mice immunized with the vaccine candidate and with strain rb51 was the number of 
 bacterial cells isolated from the mice after the challenge with virulent b. abortus strain 544 . among the 5 mice immunized ip with the 
 brucella vaccine candidate , the challenge strains were isolated from the spleens of only 3 mice . the challenge strains were isolated from the 
 spleens of only 4 mice among the 5 mice orally immunized with the vaccine candidate . in contrast , the strains were detected in spleens of all mice immunized ip 
 with strain rb51 . furthermore , in this study , mice immunized ip with the vaccine candidate showed the best protection against infection with virulent b. 
 abortus strain 544 . this observation suggests that ip immunization with the brucella vaccine candidate involves intact lps , which is a 
 potent natural immunogen . therefore , the brucella vaccine candidate elicits antibodies to lps , which are effective at protecting the animals in 
 mouse models of brucellosis .