animals and treatment : the experimental design was approved by the 
 laboratory animal care and use committee of iwate university , iwate , japan .
eight clinically 
 healthy holstein bull calves , aged 10  3 weeks , were purchased from a government farm 
 ( national livestock breeding center , morioka , japan ) and were housed in a 4  5 
 m , open - sided straw bed and naturally ventilated barn at the cattle research 
 center , iwate university .
the calves had been weaned within 4 weeks after birth and fed 
 starter pellets containing ground corn ( standard diet ) and had access to fresh water 
 ad libitum .
calves were assigned to a 4  2 experimental design for the probiotic and control groups . a 
 probiotic ( miyarisan pharmaceutical co. , ltd . ,
tokyo , japan ) , which included 
 lactobacillus plantarum strain 220 [ 9  10 colony forming 
 units ( cfu)/g ] , enterococcus faecium strain 26 ( 9  10 cfu / g ) 
 and clostridium butyricum strain miyari ( 9  10 cfu / g ) , was 
 administered daily at 3.0 g/100 kg body weight ( bw ) to the calves once daily for 5 
 consecutive days . the probiotic was mixed with 50 ml tap water in a beaker , 
 and the probiotic suspension was administered orally to each calf in a 50 
 ml drencher in the morning , prior to feeding .
calves given tap water 
 alone , with no probiotic , served as the control .
no abnormal changes in the health of any calf were observed during 
 the experimental period .
blood collection and pbmc isolation : blood was drawn by jugular 
 venipuncture from each calf at predose and on days 7 and 14 .
the first administration day 
 was considered day 1 . a blood specimen ( 6 ml ) was collected in a 
 heparinized tube ( bd vacutainer , belliver industrial estate , plymouth , u.k . ) and used for 
 pbmc isolation for flow cytometry analysis .
blood ( 4 ml ) was also collected 
 in two edta tubes ( bd vacutainer , franklin lakes , nj , u.s.a . ) and used for total rna 
 extraction from peripheral leukocytes and white blood cell ( wbc ) counts .
wbc count was 
 determined using an automatic hematology analyzer device equipped with software for bovine 
 samples ( poch-100iv diff , sysmex , kobe , japan ) .
the percentage of pbmcs was determined using 
 a hemogram method with the dif - quick staining protocol .
briefly , to purify pbmcs and to lyse 
 red blood cells , 2 ml heparinized blood was mixed with 8 
 ml 0.83% ammonium chloride solution .
after 5 min at room temperature , 
 samples were centrifuged ( 2,000 rpm , 10 min , 4c ) .
leukocytes were purified by washing twice 
 in phosphate - buffered saline ( pbs , ph 7.4 ) followed by centrifugation ( 2,000 rpm , 5 min , 
 4c ) .
after the final wash , the cell pellet was diluted in 500 l pbs , and 
 cells were counted .
then , the cell concentration was adjusted to 1  
 10/ml in pbs , and the cells were kept at 4c until used for 
 the flow cytometry assay .
flow cytometry assay : the primary monoclonal antibodies used in the 
 present study are shown in table 1table 1.monoclonal antibodies used for flow cytometry assayitems target molecule specificity mab clone isotype supplier   cd3 tcr complex pan - t cells mm1a igg1vmrd cd4 cd4 moleculet - helper cells cact183aigg1vmrd cd8 cd8cytotoxic t cells bat82aigg1vmrd cd45r ptpr pan - leukocytes gc6a igmvmrd wc1 unknown t cellsil - a29igg1vmrd cd14 lps receptor monocytes , macrophages cam36a igg1vmrd cd21 cr2 b cells baq15aigmvmrd cd282 tlr2 monocytes hca151f igg abd - serotec tcr , t cell receptor ; ptpr , protein tyrosine phosphatase receptor ; lps , 
 lipopolysaccharide ; cr2 , complement component receptor 2 ; tlr2 , toll - like receptor 
 2 .. flow cytometry analysis was performed as described previously .
briefly , pbmcs were labeled with primary antibody 
 and incubated for 60 min at 4c .
cd3 t cells and each of cd4 and 
 cd8 t cell subsets were additionally labeled with cd45r monoclonal antibody to 
 mark pan - leukocytes .
labeled cells were stained with specific purified igg conjugated to phycoerythrin ( pe ) 
 polyclonal secondary antibody ( goat anti - mouse igg1 rpe , abd serotec , kidlington , 
 oxford , u.k . ) or fluorescein isothiocyanate ( fitc)-conjugated antibody ( goat anti - mouse igg 
 or igm fitc , southern biotech , birmingham , al , u.s.a . ) and incubated for 30 min at 4c . 

flow 
 cytometry was performed using a facscan analyzer ( becton dickinson , franklin lakes , nj , 
 u.s.a . ) , equipped with a computer running the cell quest software ( becton dickinson , milan , 
 italy ) .
tcr , t cell receptor ; ptpr , protein tyrosine phosphatase receptor ; lps , 
 lipopolysaccharide ; cr2 , complement component receptor 2 ; tlr2 , toll - like receptor 
 2 .
total rna extraction from leukocytes and cdna synthesis : total rna was 
 extracted from 1 ml whole blood leukocytes using the sv total rna isolation 
 system ( promega , tokyo , japan ) .
trace genomic dna in the crude total rna samples was removed 
 by incubation with 46 units dnase i per 100 mg total rna ( ambion , austin , tx , u.s.a . ) for 
 30 min at 37c .
the concentration of total rna was verified on a 1% agarose gel , and purity 
 was determined using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies , rockland , 
 de , u.s.a . ) .
cdna was prepared using an oligo(dt ) primer and a thermal cycler ( mycycler ; 
 bio - rad , tokyo , japan ) .
real - time pcr assay : real - time pcr analysis was carried out using the iq 
 sybr green supermix kit ( bio - lad ) as described previously .
specific individual cytokine primer sequences and the internal standard gene 
 -actin used are shown in table 2table 2.primers used for real - time pcrgenegenbank access .
no.sequence ( 53 ) annealing temp ( c ) concentration ( nm ) -actin ay141970f : ggccgagcggaaatcg r : gccatctcctgctcgaagtc65750   il-6 nm173923f : gtcttcaaacgagtgggtaaag r : tgaccagaggagggaatgc65250   il-8 nm173925f : cactgtgaaaattcagaaatcattgtta r : 
 cttcacaaatacctgcacaaccttc59250   tnf- z48808 f : cggtggtgggactcgtatg r : gctggttgtcttccagcttca65 750   ifn- nm174086 f : taggcaagtctatgggatttc r : 
 gcattcattacatcatcaagtg59 250 .
amplification was carried out using a mini opticon ( bio - rad ) thermal cycler 
 with the following profile : one cycle of 95c for 2 min , followed by 45 cycles of 95c for 
 30 sec , 65 or 59c for 30 sec and 74c for 30 sec , followed by 74c for 7 min .
melting curve 
 analysis was performed from 50 to 95c , read every 0.5c holding for 5 sec .
relative gene 
 expression was calculated using the comparative threshold cycle number ( 2 ) 
 method , as described by livak and schmitge . 

results are summarized as ct values , where ct is the difference in the threshold cycle for 
 the target and -actin , as an internal control .
statistical analysis : the number of cd - positive or cd - negative cells was 
 calculated from the percentages of leukocytes gated by flow cytometry analysis and total 
 pbmcs .
5.01 ) 
 was used for statistical calculations , and one - way repeated - measures analysis of variance 
 followed by the tukey - kramer multiple comparison method was used to evaluate differences in 
 values among the groups .
subpopulations of peripheral leukocytes : the number of cd - positive cells 
 is shown in figs . 1 and 2fig.1.number of cd3 , cd4 , cd8 , cd14 , cd21 and wc1 cells in the peripheral blood 
 of calves given 3.0 g/100 kg bw of probiotic once daily for 5 consecutive days ( black 
 columns ) .
* values in the treatment group compared to the predose day in the same 
 group ( p<0.05 ) .
2.number of tlr2 ( cd282 ) cells in the peripheral blood of calves given 3.0 
 g/100 kg bw of probiotic once daily for 5 consecutive days ( black columns ) .
numbers were counted 
 at predose ( pre ) and on days 7 and 14 .
* values in 
 the treatment group compared to the predose day in the same group 
 ( p<0.05 ) .
the first administration day of the probiotic was 
 deemed day 1 .. compared to the value on the predose day and the control value on the same day , 
 increased ( p<0.05 ) numbers of cd3 t cells ( cd3 
 cd45r / cd45r ) , wc1  t cells and cd282 
 monocytes were noted in the treatment group on day 7 .
the values remained higher on day 14 ; 
 however , the difference was not statistically significant . compared to the value on the 
 predose day ,
cd8 t cells were increased ( p<0.05 ) in the 
 treatment group on day 7 .
compared to the control value on the same day , the cd4 
 t cell subset was also increased ( p<0.05 ) in the treatment group on day 
 7 .
the values of cd3 , cd4 and cd8 cell subsets decreased 
 slightly from day 7 to day 14 , and the differences were not statistically significant on day 
 14 . discrete analysis of data indicated that the numbers of cd45r and 
 cd45r t cells were increased ( p<0.05 ) in the 
 cd3 cells in the treatment group .
however , the values were increased 
 ( p<0.05 ) only in the number of cd4cd45r and 
 cd8cd45r t cells on day 7 compared to the values on the predose 
 day and/or the control ; they were not significantly different among the groups on day 14 
 ( table 3table 3.the numbers of peripheral blood mononuclear cell ( pbmc ) and t cell subpopulations 
 ( cell/l ) in probiotic - treated and control calvestreatment controlpre day 7day 14pre day 7day 14pbmc 5,283  193   6,323  3185,650  260 4,836  346 4,467  415 5,303  513 cd3cd45r1,425  117 2,199  123 * 1,940  136 1,321  157 1,431 
206 1,777  202 cd3cd45r727  60 1142  158 * 998  82 668  92 729  76 857  106 cd4cd45r191  47 315  44244  52 201  35 171  37 228  42 cd4cd45r326  62 625  79 * 394  39 328  47 305  59 333  49 cd8cd45r53  80 77  1065  12 68  13 65  28 69  13 cd8cd45r209  41 526  89 * 340  41 207  28 304  84 280  43 a ) calves received 3.0 g/100 kg of probiotic for 5 consecutive days , and additional 
 calves given the vehicle alone served as the control .
compared to the predose 
 ( pre ) values in the same group ( p<0.05 ) . #
compared to the values 
 in the control on the same day ( p<0.05 ) . ) .
the numbers of cd14 and cd21 cells changed slightly 
 in the treatment group on days 7 and 14 , but there was no statistically significant 
 difference between the treatment and control groups .
number of cd3 , cd4 , cd8 , cd14 , cd21 and wc1 cells in the peripheral blood 
 of calves given 3.0 g/100 kg bw of probiotic once daily for 5 consecutive days ( black 
 columns ) .
* values in the treatment group compared to the predose day in the same 
 group ( p<0.05 ) .
number of tlr2 ( cd282 ) cells in the peripheral blood of calves given 3.0 
 g/100 kg bw of probiotic once daily for 5 consecutive days ( black columns ) .
numbers were counted 
 at predose ( pre ) and on days 7 and 14 .
values in 
 the treatment group compared to the predose day in the same group 
 ( p<0.05 ) .
a ) calves received 3.0 g/100 kg of probiotic for 5 consecutive days , and additional 
 calves given the vehicle alone served as the control .
compared to the predose 
 ( pre ) values in the same group ( p<0.05 ) .
# compared to the values 
 in the control on the same day ( p<0.05 ) .
mrna expression of cytokines : expression of il-6 and ifn- mrna was 
 increased slightly in the treatment group on day 7 compared to the control , and the values 
 were increased ( p<0.05 ) on day 14 compared to the predose day ( fig . 3fig .
3.relative mrna expression of il-6 , il-8 , ifn- and tnf- in peripheral leukocytes of 
 calves given 3.0 g/100 kg bw of probiotic once daily for 5 consecutive days ( black 
 columns ) .
* values in the treatment group compared to the predose day in the same 
 group ( p<0.05 ) .
expression of il-8 mrna was similar between the treatment and control groups on 
 days 7 and 14 .
expression of tumor necrosis factor - alpha ( tnf- ) was slightly higher on days 
 7 and 14 in the treatment group , but the differences were not statistically significant 
 ( p=0.07 ) between the treatment and control groups .
relative mrna expression of il-6 , il-8 , ifn- and tnf- in peripheral leukocytes of 
 calves given 3.0 g/100 kg bw of probiotic once daily for 5 consecutive days ( black 
 columns ) .
* values in the treatment group compared to the predose day in the same 
 group ( p<0.05 ) .
certain probiotics have been suggested to have immune - stimulating properties in pre - weaned 
 calves and adult cattle [ 14 , 15 , 24 ] . in the present study , 
 toll - like receptor 2 ( tlr2 ; cd282 )
cell surface molecule antibody was used as a marker to 
 distinguish monocytes in peripheral blood of healthy weaned calves receiving probiotic and a 
 control group .
thus , in addition to cd282 cells , the numbers of cd3 
 t cells , cd4 , cd8 and wc1  t cell subsets and 
 cd14 and cd21 cells were evaluated .
likewise , the expression of 
 cytokine mrna , such as il-6 , il-8 , inf- and tnf- , was examined to assess the possible 
 immune - stimulating effects of the probiotic on the activation of peripheral leukocytes . in a previous study ,
the number of cd4 cells was higher after repeated 
 administration of a probiotic consisting of clostridium butyricum strain 
 miyairi in dairy cows . in another study , 

cd8 cells were stimulated in the peripheral blood of pre - weaned calves by 
 administration of a bacteria - based probiotic . 

furthermore , a number of in vitro and in vivo studies on 
 human pbmcs have suggested that a probiotic containing e. 
 faecium , c. butyricum or 
 lactobacillus subspecies shows enhancing effects on t cell subsets and 
 monocytes [ 12 , 35 ] . in young mammals , most cd3
, cd4 and cd8 
 cells express the high - molecular - weight isoform of cd45r , and an increase in the number of t lymphocytes expressing cd45r facilitates 
 regular activation of t cells . in the present 
 study , the increase in the number of cd3cd45r t cells in the 
 treatment group , compared to the predose day or control , may indicate a nonspecific , but 
 enhanced immune state with possibly multiple causes after the 5-day repeated administration 
 of the probiotic .
similar to cd3 cells , increased numbers of 
 cd4cd45r and cd8cd45r t cells were 
 observed .
they may be involved in immune system responses , along with direct or indirect 
 immune - stimulatory effects of the probiotic in the calves , as suggested by previous studies 
 in cattle [ 6 , 15 , 24 ] .
the wc1 molecule is a member of the scavenger receptor cysteine - rich ( srcr ) family found on 
  t cells , which are negative for cd4 and cd8 . 
 in cattle ,
the regulatory potential of wc1  t cell subsets is comparable to 
 that of cd14 cells , and the 
 wc1 t cell subpopulation is larger in calves .
bovine  lymphocyte subsets are activated indirectly through cytokines 
 secreted by t cells , antigen - presenting cells ( apcs ) and other accessory cells .
as our results show , the higher number of 
 wc1  t cells in the calves receiving the probiotic was associated with an 
 increased number of cd4 cells , which might suggest stimulation of cellular 
 immunity in the calves .
although wc1  t cells and cd14 cells are 
 generated comparably in bovines , we found that 
 there were more wc1 t cells than cd14 cells . on the other hand , 

wc1 cells are immune suppressors , secreting ifn- in bovines [ 11 , 27 ] , 
 consistent with our results .
the expression of tlrs has been correlated with the population of total bacteria and lab in 
 the gastrointestinal tract , including the rumen of calves , during the first 6 months of life 
 . moreover ,
the expression of tlr2 was evident 
 on bovine antigen presenting cells with high level expression on peripheral blood monocytes 
 and monocyte - derived macrophages .
the tlr2 cell 
 surface molecule on bovine monocyte - derived dendritic cells ( dcs ) and gastrointestinal 
 macrophages is most responsible for recognition of gram - positive bacteria [ 4 , 9 , 10 ] .
mechanisms indicating a direct role of tlr2 include 
 bacterial reorganization by epithelial macrophages in calves .
the transcytosis processing of bacterial particles via ileal peyer s 
 patch m cells and the later appearance of tlrs on dcs in the lamina propria play a key role 
 in the mechanism of probiotic immunomodulation in cattle [ 7 , 20 , 21 ] .
thus , dcs are the principal stimulators of t cells [ 7 , 12 ] .
the tlr signaling pathway 
 is involved with the actions of a probiotic containing c. 
 butyricum , and the absence of tlr2 expression might indicate the need for 
 tlr2 in the recognition of clostridium bacteria [ 9 , 10 ] . in the present study
, we 
 provide evidence for an increased number of tlr2 monocytes in the peripheral 
 blood of calves receiving a probiotic consisting of three species of gram - positive bacteria , 
 including c. butyricum .
the increased number of peripheral 
 monocytes expressing tlr2 might be associated with the formal reaction of innate immunity in 
 response to increasing and changing bacterial diversity in the gastrointestinal tract of the 
 calves receiving the probiotic .
furthermore , the cd14 cell surface molecule is essential for 
 the response to most gram - negative bacteria , including lipopolysaccharides , via the tlr4 and 
 md2 complex [ 22 , 28 ] .
the tlr2 molecule recognizes microbial - associated molecule patterns 
 independently [ 4 , 7 ] .
although some differences were noted between the different antibodies assessed 
 the expression of tlr2 and cd14 monocytes in bovine .
moreover , tlr2 is expressed on peripheral blood b lymphocytes ; however , a b cell receptor complex including the cd21 
 molecule is present on mature b cells and also ileal peyer s patch - derived b lymphocytes in 
 healthy calves . similar to these reports ,
the 
 numbers of cd14 and cd21 b cells in the present study were slightly 
 increased in the calves on days 7 and 14 .
probiotics including lactobacillus and c. 
 butyricum induce the release of proinflammatory cytokines , such as il-6 
 and tnf- , which are involved in nonspecific immune responses [ 9 , 10 ] .
a probiotic consisting of 
 e. faecium and c. 
 butyricum upregulates the level of ifn- and downregulates tnf- in the 
 supernatant of human pbmcs . in our study , 

increased expression of il-6 , inf- and tnf- mrna was observed in the treatment group on 
 days 7 and 14 .
increased expression of il-6 , ifn- and tnf- in peripheral leukocytes may be 
 dependent on the period after administration and numbers of activated t lymphocytes or 
 monocytes in the treatment group .
, in which increased 
 expression of ifn- was observed only 2 weeks after trickle stimulation of cultured pbmcs 
 from calves . on the other hand , expression of il-8 is related to neutrophil function , 
 especially inflammatory stimulation , in bovine . in 
 the present study ,
the expression of il-8 was unchanged compared to the predose day and the 
 control value on the same day , likely because the calves were healthy . in conclusion ,
repeated 5-day administration of a bacteria - based probiotic resulted in 
 increased numbers of cd3 t cells , cd4 , cd8 and 
 wc1 t cell subsets and cd282 monocytes in healthy holstein calves . 
 moreover , higher mrna expression of pro - inflammatory cytokines , such as il-6 , inf- and 
 tnf- , in peripheral leukocytes was noted .
these results suggest that the peripheral blood 
 leukocytes and cellular immune function in the healthy calves may have been stimulated via 
 effects on the gastrointestinal microbiota , which generally increased , with overall 
 stability of the rumen bacterial flora , by the oral probiotic treatment .
 abstracteight holstein calves ( 10  3 weeks ) were used to examine the interaction between a 
 bacteria - based probiotic agent ( probiotic ) and the function of peripheral blood 
 mononuclear cells ( pbmcs ) . 
 the probiotic , consisting of lactobacillus 
 plantarum , enterococcus faecium and clostridium 
 butyricum , was administered orally at 3.0 g/100 kg body weight to calves once 
 daily for 5 consecutive days . 
 calves given the vehicle alone with no probiotic served as 
 the control . 
 in the treatment group , increases in numbers of cd282 + ( tlr2 ) 
 monocytes , cd3 + t cells and cd4 + , cd8 + and 
 wc1 +  t cell subsets were noted on day 7 post - placement compared to predose 
 day and the control group . 
 expression of interleukin ( il)-6 , interferon - gamma ( inf- ) and 
 tumor necrosis factor - alpha ( tnf- ) was elevated in peripheral leukocytes on days 7 and 
 14 . 
 these results suggest that peripheral blood leukocytes in healthy calves may be 
 stimulated via the gastrointestinal microbiota , which was increased by the oral probiotic 
 treatment , with overall stability of the rumen bacterial flora . 
 the 5-day repeated 
 administration of a bacteria - based probiotic may enhance cellular immune function in 
 weaned calves . 
MATERIALS AND METHODS
RESULTS
DISCUSSION