source and preparation of hbv for spray administration : the hbv was 
 provided by wissen co. , ltd . it is formulated fractions purified from the 
 bee venom obtained by large quantiti bee - venom collector ( p10 - 1003672 , wissen co. , 
 ltd . ) . the fine hbv powder was dissolved in a solvent consisting of 95.7% distilled water , 3.5% 
 ethanol and 0.8% propylene glycol , by volume , for spray administration . its concentration 
 was 2.1 mg / ml , which was the optimal concentration determined in our 
 preliminary experiments . chicks and administration of hbv spray : one - day - old ross broiler chicks 
 with no history of salmonellosis were obtained from a local hatchery . after a 2-day 
 acclimation period , the chicks were randomly divided into two groups consisting of 60 chicks 
 each . the experimental group 
 ( hbv - sprayed group ) was exposed to the hbv spray in solvent . as the chicks grew , 30 , 60 and 
 90 ml solvent volumes were administered to each group at 3 , 9 and 15 days 
 of age , respectively . after three sprays , chicks were subjected to three independent 
 experiments including an evaluation of antibody production against formalin - killed 
 s. gallinarum ( experiment 1 ) , an evaluation of immune response and 
 bacterial clearance against a sublethal s. gallinarum dose [ 1  
 10 colony forming units ( cfu ) ] ( experiment 2 ) and an evaluation of 
 survivability and pathological changes against a low lethal dose ( ld25 , 5  
 10 cfu ) of the bacteria ( experiment 3 ) . all birds were housed in separate 
 air - controlled rooms by group and allowed free access to nutritionally complete 
 antibiotic - free chick feed and tap water . all animal procedures were approved by the 
 institutional animal care and use committee of chonnam national university ( approval number : 
 cnu iacuc - yb-2010 - 1 ) . experiment 1evaluation of antibody production activity against formalin - killed s. 
 gallinarum : on day 1 after the last spray , 10 chicks were randomly selected from 
 each group and isolated in separate rooms by group . in this experiment , 1  10 
 cfu / ml formalin - killed s. gallinarum ( sg3001 ) was used 
 as the antigen . all chicks received two subcutaneous injections ( 0.3 ml 
 each time ) with a 1-week interval between injections . the first inoculation was given with 
 freund s complete adjuvant ( sigma - aldrich , st . louis , mo , u.s.a . ) at 16 days of age , and the 
 second was given with freund s incomplete adjuvant ( sigma - aldrich ) at 23 days of age . one was 
 collected before immunization , and the other was collected 1 week after the last 
 immunization . serum was obtained by centrifugation at 2,000  g for 10 min 
 at 4c , and the serum was inactivated at 56c for 30 min . antibody production was measured 
 by enzyme - linked immunosorbent assay ( elisa ) , as described previously . briefly , 96-well plates ( iwaki , tokyo , japan ) were coated with 100 
 l of a solution containing 20 g whole formalin - killed 
 s. gallinarum ( used as antigen ) in 1 ml of 0.1 m 
 carbonate - bicarbonate buffer ( ph 9.6 ) and left overnight at 4c . after three washes with pbs 
 containing 0.05% tween 20 ( pbs - t ) , the wells were saturated with 200 l of 
 5% skim milk ( bd biosciences , franklin lakes , nj , u.s.a . ) . the wells were washed three times 
 with pbs - t after a 2 hr incubation at room temperature . the diluted samples were added to wells and incubated at room 
 temperature for 1 hr . after three washes with pbs - t , 100 l of a 1:5,000 
 dilution of horseradish peroxidase - conjugated rabbit anti - chicken igy ( jackson 
 immunoresearch , west grove , pa , u.s.a . ) was added to each well . after a 1 hr incubation at 
 room temperature , the plates were washed , and 100 l of a substrate 
 consisting of 0.05 m citrate buffer ( ph 4.0 ) , 
 2 - 2-azino - bis(3-ethylbenzthiazoline-6-sulfonic acid ) ( bio basic , markham , on , canada ) and 
 30% hydrogen peroxide was added to each well . after a 10 min incubation at room temperature 
 in the dark , the reaction was stopped with 5% sodium dodecyl sulfate ( applichem , darmstadt , 
 germany ) . reactions were read at an optical density of 405 nm using an elisa plate reader 
 ( thermo labsystems , helsinki , finland ) . pre - immunized chick serum was used as a negative 
 control , and each sample was tested in duplicate . experiment 2evaluation of immune responses and bacterial clearance against a 
 sublethal dose of s. gallinarum : thirty chicks were randomly selected from each 
 group and isolated in separate rooms by group at 1 day after the last spray administration . ten chicks from each isolated group were randomly sacrificed for analysis of the immune 
 response in non - infected chicks , and the remaining 20 chicks in each isolated group were 
 orally inoculated with 1  10 cfu ( sublethal dose ) s. 
 gallinarum ( sg3001 ) . five infected chicks from each isolated group were randomly sacrificed 
 for analysis of the immune response and bacterial clearance against a sublethal dose of 
 s. gallinarum at 3 , 7 , 10 and 14 days post - infection ( dpi ) . experiment 2.1determination of t lymphocyte subpopulation in the spleen : 
 the spleen was obtained from each sacrificed chick at 0 dpi ( the day of infection but before 
 infection ) and at 3 , 7 , 10 and 14 dpi , and single - cell suspensions were prepared by forcing 
 the tissue through 40-m nylon mesh ( bd biosciences ) . isolated cells were 
 analyzed to determine cd4cd8 t lymphocyte and 
 cd4cd8 t lymphocyte percentages , as described previously . the cells were stained with both fluorescein 
 isothiocyanate ( fitc)-conjugated mouse anti - chicken cd4 ( southern biotech , birmingham , al , 
 u.s.a . ) and phycoerythrin ( pe)-conjugated mouse anti - chicken cd8 ( southern biotech ) . after a 
 30 min incubation at room temperature in the dark , the cells were washed twice with pbs , and 
 the lymphocyte subpopulations were analyzed using a facsort flow cytometer ( bd biosciences ) . viable lymphocytes were gated by forward and side - scatter characteristics ( fsc / ssc ) , and 
 10,000 events were analyzed for fitc or pe positive staining . results of each lymphocyte 
 subpopulation are expressed as percentages of events in the fsc / ssc lymphocyte gate . experiment 2.2evaluation of relative il-18 and ifn- mrna expression levels in 
 spleen : total rna was extracted from splenocytes using the rneasy mini kit 
 ( qiagen , valencia , ca , u.s.a . ) , and target rna was reverse transcribed using the 
 quantitect reverse transcription kit ( qiagen ) , according to the manufacturer s 
 instructions . the chicken il-18 , ifn- and gapdh primer sets were designed as 
 described previously , and their sequences are 
 shown in table 1table 1.primer used for the relative real - time polymerase chain reaction ( pcr)nucleotide sequence ( 53)accession number il-18fwaggtgaaatctggcagtggaatnm_204608rvtgaaggcgcggtggtttifn-fwgctcccgatgaacgacttganm_205149rvtgtaagatgctgaagagttcattcggapdhfwcctaggatacacagaggaccaggttnm_204305rvggtggaggaatggctgtcafw , forward primer ; rv , reverse primer ; il-18 , interleukin-18 ; ifn- , 
 interferon - gamma ; gapdh , glyceraldehyde-3-phosphate dehydrogenase . a ) cdna genbank 
 accession number and corresponding genes are available online at 
 http://www.ncbi.nlm.nih.gov/ .. il-18 and ifn- mrna levels were determined by real - time polymerase chain 
 reaction ( pcr ) assay using myiq2 ( bio - rad laboratories , hercules , ca , u.s.a . ) 
 with the iq sybr green supermix ( bio - rad laboratories ) , according 
 to the manufacturer s instructions . each sample was amplified in duplicate in the same 
 96-well pcr plate , and each procedure was repeated twice . the relative quantitative pcr 
 conditions were 5 min at 95c followed by 45 cycles of 30 sec at 94c , 30 sec at 57c and 45 
 sec at 72c . a melting program was run after amplification to verify the presence of only 
 one pcr product . the threshold cycle ( ct ; the cycle number at which the amount of amplified 
 gene of interest reached a fixed threshold ) was determined subsequently . relative 
 quantification of il-18 and ifn- mrna expression was calculated by the comparative ct 
 method , as described previously . the relative 
 quantification values of the target genes ( il-18 and ifn- ) were normalized to an endogenous 
 control gene ( gapdh ) and relative to a calibrator . the values were expressed as 
 2(fold ) , where ct = ct of target gene  ct of endogenous control gene and 
 ct=ct of samples for target gene  ct of the target gene calibrator . fw , forward primer ; rv , reverse primer ; il-18 , interleukin-18 ; ifn- , 
 interferon - gamma ; gapdh , glyceraldehyde-3-phosphate dehydrogenase . a ) cdna genbank 
 accession number and corresponding genes are available online at 
 http://www.ncbi.nlm.nih.gov/. experiment 2.3determination of serum lysozyme activity : blood was 
 collected from the wing vein of each sacrificed chick at 0 , 3 , 7 , 10 and 14 dpi . serum was 
 obtained by centrifugation at 2,000  g for 10 min at 4c and used to 
 determine lysozyme activity . briefly , a standard 
 dilution series was produced by dissolving crystalline lysozyme ( sigma - aldrich ) in a 
 phosphate buffer ( ph 6.2 ) to concentrations of 1 , 2 , 3 , 4 , 5 or 6 
 g / ml . twenty microliters of each solution was added to 
 two wells of a 96-well microtiter plate . two hundred microliters of phosphate buffer was 
 added to one well , and 200 l of a micrococcus 
 lysodeikticus solution ( 600 mg / l phosphate buffer ; 
 sigma - aldrich ) was added to the other well . the absorbance was determined at 540 nm after 
 15 , 30 , 45 and 60 min incubations at 41c . the regression coefficient ( b ) 
 between absorbance and time was calculated for each concentration . lysozyme activity ( lysis of 
 m. lysodeikticus ) was determined based on b and the 
 lysozyme concentration . experiment 2.4viable bacterial counts in liver , the bursa of fabricius and 
 cecum : viable bacteria cell counts were carried out for the liver , bursa of 
 fabricius and cecum collected from each sacrificed chick at 3 , 7 , 10 and 14 dpi as follows . a 10% ( w / v ) suspension of each tissue sample was prepared in sterile pbs . the pool was 
 homogenized using a precellys24 ( bertin technologies , montigny - le - bretonneux , 
 france ) . one hundred microliters of the homogenate was serially diluted 10-fold in pbs , and 
 100 l of each dilution was spread onto a xylose lysine deoxycholate agar 
 plate and incubated at 37c for 24 hr . characteristic black - colored colonies were counted 
 and expressed as log cfu / g tissue , but only for those colonies with counts of 30300 per 
 plate . in addition , representative colonies were subjected to gram staining and biochemical 
 tests for identification . experiment 3evaluation of survivability and pathological changes against a low 
 lethal dose of s. gallinarum : twenty chicks were randomly selected from each 
 group and isolated in separate rooms by group at 1 day after the last spray administration . all chicks were orally inoculated with 5  10 cfu ( ld25 ) 
 s. gallinarum ( sg3001 ) . necropsy 
 examinations were carried out on all dead and euthanized chicks , and any pathological 
 changes associated with fowl typhoid were observed . . 
 student s t - test was performed for the statistical analysis of all data . all statistical analyses were performed using sigmaplot version 10.0 software 
 ( systat software , san jose , ca , u.s.a . ) . body weight changes : body weight was recorded at 3 , 9 and 15 days of age 
 in all chicks and at 23 ( 7 dpi ) and 33 ( 17 dpi ) days of age in surviving chicks infected 
 with a low lethal dose of s. gallinarum ( fig . 1fig . 1.effect of honeybee venom ( hbv ) on body weight changes in broiler chicks . body weight 
 was recorded at 3 , 9 and 15 days of age in all chicks and was also recorded at 23 and 
 33 days of age in surviving chicks after infection with a low lethal dose of 
 salmonella gallinarum . data are mean  
 standard deviation ( 315 days of age : n=60/group , 23 days of age : control group and 
 hbv - sprayed group : n=17 and 19 , respectively , 33 days of age : control group and 
 hbv - sprayed group : n=15 and 17 , respectively ) . * p<0.05 
 vs. control , * * * p<0.001 vs. 
 control . ) . the initial chick body weight did not differ between the groups at 3 days of age 
 ( control group and hbv - sprayed group : 62.0  2.4 and 62.1  2.4 g , respectively ) . however , 
 the hbv - sprayed group showed a tendency for increasing body weight compared with that in the 
 control group at 9 and 15 days of age , but the difference was not significant 
 ( p=0.060 and 0.053 at 9 and 15 days of age , respectively ) . this increase 
 in body weight reached significance at 23 ( p<0.05 ) and 33 days of age 
 ( p<0.001 ) . body weight 
 was recorded at 3 , 9 and 15 days of age in all chicks and was also recorded at 23 and 
 33 days of age in surviving chicks after infection with a low lethal dose of 
 salmonella gallinarum . data are mean  
 standard deviation ( 315 days of age : n=60/group , 23 days of age : control group and 
 hbv - sprayed group : n=17 and 19 , respectively , 33 days of age : control group and 
 hbv - sprayed group : n=15 and 17 , respectively ) . experiment 1antibody production activity against formalin - killed s. 
 gallinarum : antibody production activity ( determined at od405 nm ) 
 against formalin - killed s. gallinarum in the hbv - sprayed group ( 1.97  
 0.26 ) increased significantly compared to that in the control group ( 1.67  0.27 ; 
 p<0.05 ) ( fig . 2fig . 2.effect of honeybee venom ( hbv ) on antibody production activity in broiler chicks . serum antibody production was determined 
 ( od405 nm ) using an enzyme - linked immunosorbent assay plate reader . serum 
 antibody production levels in the hbv - sprayed group were significantly higher compared 
 to those in the control group . sera of pre - immunized chicks displayed a clear negative reaction ( control group and 
 hbv - sprayed group : 0.35  0.10 and 0.34  0.08 , respectively ) against the 
 s. gallinarum antigen . effect of honeybee venom ( hbv ) on antibody production activity in broiler chicks . serum antibody production was determined 
 ( od405 nm ) using an enzyme - linked immunosorbent assay plate reader . serum 
 antibody production levels in the hbv - sprayed group were significantly higher compared 
 to those in the control group . experiment 2.1t lymphocyte subpopulation in spleen : the percentage of 
 cd4cd8 t lymphocytes in the spleen of the hbv - sprayed group tended 
 to increase during the entire experimental period compared with that in the control group 
 ( fig . 3afig . 3.effect of honeybee venom ( hbv ) on t lymphocyte subpopulations in the spleen of 
 broiler chicks . ( a ) the percentage of cd4cd8 t lymphocyte in 
 the spleen of the hbv - sprayed group tended to increase compared with that in the 
 control group . ( b ) conversely , the percentage of cd4cd8 t 
 lymphocytes in spleen of the hbv - sprayed group tended to decrease compared with that 
 in the control group . ( c ) consequently , the cd4:cd8 ratio in 
 the spleen of the hbv - sprayed group tended to increase during the entire experimental 
 period compared with that in the control group . 0 days post - infection ( dpi ) is the day 
 of infection , but before infection . data are mean  standard deviation ( 0 dpi : n=10/group , 314 dpi : n=5/group ) . in particular , the difference in the percentage of cd4cd8 t 
 lymphocytes was significant between the groups at 14 dpi ( p<0.05 ) . in 
 contrast , the percentage of cd4cd8 t lymphocytes in the spleen of 
 the hbv - sprayed group tended to decrease during the entire experimental period compared with 
 that in the control group ( fig . the 
 difference in the percentage of cd4cd8 t lymphocyte was significant 
 between the groups at 0 dpi ( p<0.01 ) . consequently , the 
 cd4:cd8 ratio in the spleen of the hbv - sprayed group tended to 
 increase during the entire experimental period compared with that in the control group 
 ( fig . 3c ) . in particular , the difference in the 
 cd4:cd8 cell ratio was significant between the groups at 0 and 14 
 dpi ( p<0.05 ) . effect of honeybee venom ( hbv ) on t lymphocyte subpopulations in the spleen of 
 broiler chicks . ( a ) the percentage of cd4cd8 t lymphocyte in 
 the spleen of the hbv - sprayed group tended to increase compared with that in the 
 control group . ( b ) conversely , the percentage of cd4cd8 t 
 lymphocytes in spleen of the hbv - sprayed group tended to decrease compared with that 
 in the control group . ( c ) consequently , the cd4:cd8 ratio in 
 the spleen of the hbv - sprayed group tended to increase during the entire experimental 
 period compared with that in the control group . 0 days post - infection ( dpi ) data are mean  standard deviation ( 0 dpi : n=10/group , 314 dpi : n=5/group ) . experiment 2.2relative il-18 and ifn- mrna expression levels : the 
 relative il-18 and ifn- mrna expression levels in spleen of the hbv - sprayed group tended to 
 increase during the entire experimental period compared with those in the control group 
 ( fig . 4.effect of honeybee venom ( hbv ) on relative interleukin ( il)-18 and interferon ( ifn)- 
 mrna expression levels in the spleen of broiler chicks . the relative mrna expression 
 levels of il-18 ( a ) and ifn- ( b ) in spleen of the hbv - sprayed group tended to 
 increase during the entire experimental period compared with those in the control 
 group . 0 days post - infection ( dpi ) is the day of infection , but before infection . data are mean  standard deviation ( 0 
 dpi : n=10/group , 314 dpi : n=5/group ) . notably , the relative ifn- mrna expression levels in spleen of the hbv - sprayed 
 group increased significantly compared with those in the control group at 10 
 ( p<0.01 ) and 14 dpi ( p<0.05 ) . effect of honeybee venom ( hbv ) on relative interleukin ( il)-18 and interferon ( ifn)- 
 mrna expression levels in the spleen of broiler chicks . the relative mrna expression 
 levels of il-18 ( a ) and ifn- ( b ) in spleen of the hbv - sprayed group tended to 
 increase during the entire experimental period compared with those in the control 
 group . 0 days post - infection ( dpi ) is the day of infection , but before infection . data are mean  standard deviation ( 0 
 dpi : n=10/group , 314 dpi : n=5/group ) . experiment 2.3serum lysozyme activity : serum lysozyme concentration in 
 the hbv - sprayed group ( 2.47  0.35 g / ml ) was 
 significantly higher than that in the control group ( 1.45  0.22 
 g / ml ) before the experimental s. 
 gallinarum infection ( p<0.001 ) . moreover , serum lysozyme concentration 
 in the hbv - sprayed group tended to increase during the entire experimental infection period 
 compared with that in the control group , although the difference was not significant ( fig . 5fig . serum 
 lysozyme concentration in the hbv - sprayed group was significantly higher than that in 
 the control group before the experimental salmonella gallinarum 
 infection ( 0 days post infection [ dpi ] ) . moreover , serum lysozyme concentration in the 
 hbv - sprayed group tended to increase during the entire experimental infection period 
 compared with that in the control group . data are mean  sd ( 0 dpi : 
 n=10/group , 314 dpi : n=5/group ) . * * * p<0.001 vs. 
 control . ) . serum 
 lysozyme concentration in the hbv - sprayed group was significantly higher than that in 
 the control group before the experimental salmonella gallinarum 
 infection ( 0 days post infection [ dpi ] ) . moreover , serum lysozyme concentration in the 
 hbv - sprayed group tended to increase during the entire experimental infection period 
 compared with that in the control group . 0 data are mean  sd ( 0 dpi : 
 n=10/group , 314 dpi : n=5/group ) . * * * p<0.001 vs. 
 control . experiment 2.4bacterial clearance in chicks infected with a sublethal dose of s. 
 gallinarum : viable bacterial cells in the liver , bursa of fabricius and cecum of 
 chicks in both groups were maximum at 7 dpi , after which there was a moderate decline until 
 14 dpi ( fig . 6.effect of honeybee venom ( hbv ) on bacterial clearance in chicks infected with a 
 sublethal dose of salmonella gallinarum . viable bacteria cell counts 
 were carried out in the liver ( a ) , bursa of fabricius ( b ) and cecum ( c ) collected from 
 each sacrificed chick at 3 , 7 , 10 and 14 days post - infection ( dpi ) . the number of viable bacterial cells in those tissues in the hbv - sprayed group 
 tended to decrease during the entire experimental infection period compared with that in the 
 control group . differences in the viable bacterial cell counts were significant at 3 dpi 
 ( p<0.05 ) in the liver ( fig . 6a ) , at 3 ( p<0.01 ) , 7 ( p<0.01 ) and 10 dpi 
 ( p<0.05 ) in the bursa of fabricius ( fig . 6b ) and at 7 ( p<0.01 ) and 14 dpi 
 ( p<0.05 ) in the cecum ( fig . effect of honeybee venom ( hbv ) on bacterial clearance in chicks infected with a 
 sublethal dose of salmonella gallinarum . viable bacteria cell counts 
 were carried out in the liver ( a ) , bursa of fabricius ( b ) and cecum ( c ) collected from 
 each sacrificed chick at 3 , 7 , 10 and 14 days post - infection ( dpi ) . experiment 3.1survivability against a low lethal dose of s. gallinarum : 
 mortality was first observed at 4 dpi in the control group , whereas mortality in the 
 hbv - sprayed group was delayed for 3 days compared with that in the control group . survival 
 rates in the hbv - sprayed group remained higher throughout the experimental infection period 
 than those in the control group . survival rates were 75% ( 15 of 20 chicks ) in the control 
 group and 85% ( 17 of 20 chicks ) in the hbv - sprayed group by 17 dpi ( fig . 7fig . 7.survival rate trends in chicks infected with a low lethal dose of 
 salmonella gallinarum . mortality was first observed at 4 days 
 post - infection ( dpi ) in the control group , whereas mortality in the honey bee venom 
 ( hbv)-sprayed group was delayed for 3 days compared with that in the control group . survival rates in the hbv - sprayed group remained higher throughout the experimental 
 infection period than those in the control group . survival rates were 75% ( 15 of 20 
 chicks ) in the control group and 85% ( 17 of 20 chicks ) in the hbv - sprayed group by 17 
 dpi . survival rate trends in chicks infected with a low lethal dose of 
 salmonella gallinarum . mortality was first observed at 4 days 
 post - infection ( dpi ) in the control group , whereas mortality in the honey bee venom 
 ( hbv)-sprayed group was delayed for 3 days compared with that in the control group . survival rates in the hbv - sprayed group remained higher throughout the experimental 
 infection period than those in the control group . survival rates were 75% ( 15 of 20 
 chicks ) in the control group and 85% ( 17 of 20 chicks ) in the hbv - sprayed group by 17 
 dpi . experiment 3.2pathological changes in chicks infected with a low lethal dose of s. 
 gallinarum : chicks infected with a low lethal dose of s. 
 gallinarum displayed typical gross lesions of fowl typhoid at necropsy . congested 
 subcutaneous blood vessels and dark brown skeletal muscles were observed in septicemic 
 carcasses . petechial hemorrhaging and necrotic foci were common features in the liver , 
 spleen and myocardium . in addition , hepatosplenomegaly , hydropericardium , nephropathy and 
 intestinal tract congestion were also observed . although these pathological changes were 
 observed in both groups , the control group showed more severe petechial hemorrhaging and 
 necrotic foci in the liver ( fig . 8afig . the control group showed more severe 
 petechial hemorrhaging and necrotic foci in the liver ( a ) and also displayed more 
 severe fibrinous inflammation in the liver and hydropericardium as well as intestinal 
 tract congestion ( b ) compared with those in the honeybee venom ( hbv)-sprayed group 
 ( c ) . ) and also displayed more severe fibrinous inflammation in the liver , hydropericardium 
 and intestinal tract congestion ( fig . 8c ) . gross findings in chicks infected with a low lethal dose of 
 salmonella gallinarum . the control group showed more severe 
 petechial hemorrhaging and necrotic foci in the liver ( a ) and also displayed more 
 severe fibrinous inflammation in the liver and hydropericardium as well as intestinal 
 tract congestion ( b ) compared with those in the honeybee venom ( hbv)-sprayed group 
 ( c ) . antibiotics are used as feed or drinking water additives to improve growth performance and 
 to prevent subclinical disease challenge in the poultry industry . therefore , the present study monitored body weight changes and 
 evaluated whether hbv is an effective immunostimulant that can protect against 
 s. gallinarum , which remains a significant problem in many countries 
 [ 21 , 22 ] . our results demonstrated that administering hbv by spray had a beneficial effect on body 
 weight gain in broiler chicks . this finding was similar to previous reports showing that 
 subcutaneous injections of hbv and acupuncture increase body weight gain in young pigs 
 , and hbv supplementation via drinking water 
 improves body weight gain and feed intake in broiler chicks . notably , we showed that the increase in body weight following hbv spray 
 administration was more pronounced in broiler chicks infected with a low lethal dose of 
 s. gallinarum . these results suggest that administering hbv by spray 
 could help improve growth performance in the absence of infection as well as in environments 
 with a risk of exposure to contaminating pathogens , such as conventional farms . antibody production against formalin - killed s. gallinarum increased 
 significantly in the hbv - sprayed group compared with that in the control group . this finding 
 was similar to a previous report that acupuncture with natural honey bee stings enhances 
 antibody production against the classical swine fever virus vaccine , mycoplasma 
 hyopneumoniae vaccine and the atrophic rhinitis vaccine in pigs . therefore , our result indicates that humoral 
 immunity was enhanced by administering hbv to broiler chicks . low cd4:cd8 ratios are usually observed in individuals 
 with acute viral diseases and hemophilia , whereas 
 high ratios have been associated with an increase in chick immunofunctional ability [ 1 , 16 , 17 ] . we showed that the cd4:cd8 
 ratio in the spleen of the hbv - sprayed group increased during the entire experimental period 
 compared with that in the control group , suggesting that the hbv spray administration 
 conferred a benefit on broiler chick immune function . this finding echoed that of nam 
 et al . , who found that an 
 intraperitoneal injection of hbv increases the percentage of cd4cd8 
 t lymphocytes and decreases the percentage of cd4cd8 t lymphocytes 
 in the spleen of balb / c mice . the relative il-18 and ifn- mrna expression levels in spleen of the hbv - sprayed group 
 tended to increase during the entire experimental period compared with those in the control 
 group . of note , the enhanced relative ifn- expression following administration of the hbv 
 spray was significant in broiler chicks infected with a sublethal dose of 
 s. gallinarum . this observation agreed with the finding of nam et 
 al . , who noted that an 
 intraperitoneal injection of hbv enhances ifn- expression in balb / c mice . il-18 and ifn- 
 are mainly produced by activated macrophages and stimulated cd4 t lymphocytes 
 [ 7 , 29 ] . these cytokines play an important role in the host defense against infection by microbial 
 pathogens and induce a variety of physiologically significant responses that contribute to 
 immunity [ 7 , 29 ] . lysozyme , which is secreted by some phagocytes , such as macrophages and polymorphonuclear 
 leukocytes , is highly active against bacteria . lysozyme destroys glucosidic bonds in the 
 cell walls of escherichia coli and staphylococcus as a 
 result of its phagocytic activity . therefore , high 
 serum lysozyme activity is associated with high phagocyte destructive activity . in the present study , the serum lysozyme 
 concentration in the hbv - sprayed group was significantly higher than that in the control 
 group before the experimental s. gallinarum infection . moreover , serum 
 lysozyme concentration in the hbv - sprayed group tended to increase during the entire 
 experimental infection period compared to that in the control group . the number of viable bacterial cells in all tested tissues of the hbv - sprayed group tended 
 to decrease compared with that in the control group after infection with a sublethal dose of 
 s. gallinarum . moreover , mortality in the hbv - sprayed group was delayed 3 
 days compared with that in the control group , and the final survival rate in the hbv - sprayed 
 group was 10% higher than that in the control group after the experimental infection with a 
 low lethal dose of s. gallinarum . additionally , the necropsy revealed that 
 the hbv - sprayed group showed mild and less severe abnormal gross lesion pathological changes 
 compared with those in the control group , indicating that the hbv spray reinforced bacterial 
 clearance and increased survivability against s. gallinarum . previous 
 studies have shown that improving the general immune status of birds through prophylactic 
 administration of natural immunostimulants helps alleviate clinical symptoms and increases 
 survivability against s. gallinarum [ 16 , 17 ] . therefore , it can be presumed that 
 the prophylactic effects of administering the hbv against s. gallinarum are 
 also associated with stimulation of the non - specific immune response . taken together , our findings suggest that the hbv spray improved body weight gain , 
 particularly in the presence of infection and enhanced protective immune activity against 
 s. gallinarum . hence , the hbv spray may provide an alternative way to 
 decrease antibiotic use , particularly in chicks . however , the exact mechanisms of the hbv 
 spray on body weight gain and bacterial clearance were not determined . therefore , these 
 mechanisms of the hbv spray must be assessed in a future study . in addition , confirmation of 
 the protective efficacy of the hbv spray is required in naturally occurring fowl typhoid .
abstractantibiotics continue to be used as growth promoters in the poultry industry . 
 honeybee 
 ( apis melifera ) venom ( hbv ) possesses a number of beneficial biological 
 activities , particularly for regulating the immune system . 
 the aim of the present study 
 was to evaluate the immunoprophylactic effects of hbv against salmonella 
 gallinarum in broiler chicks as an initial step towards developing eco - friendly 
 alternatives to reduce antibiotic use . 
 hbv was administered using a spray technique . 
 hbv 
 improved body weight gain , particularly in the presence of infection . 
 moreover , hbv 
 enhanced antibody production activity against formalin - killed s. 
 gallinarum . the cd4+:cd8 + t lymphocyte ratio , relative mrna 
 expression levels of interleukin-18 and interferon- , and serum lysozyme activity also 
 increased following hbv administration before the infection period as well as during 
 infection . 
 hbv reinforced bacterial clearance and increased survivability against 
 s. gallinarum . 
 corresponding pathological analyses demonstrated that 
 the hbv - sprayed group displayed mild and less severe abnormal changes compared with those 
 in the control group . 
 it was presumed that the prophylactic effects of hbv against 
 s. gallinarum were associated with its non - specific immune response 
 stimulating activity . 
 thus , hbv may provide an alternative to reduce antibiotic use in the 
 poultry industry .