animals and treatment : the experimental design was approved by the 
 laboratory animal care and use committee of iwate university , iwate , japan .
forty - two 
 scouring and healthy pre - weaning holstein calves ( age 10  5 days ) housed in two commercial 
 dairy farms were used in the experiment .
all calves were examined clinically , and the 
 scouring status of each calf was inspected .
the scour condition was watery to muddy yellow , 
 and the appetite of the scouring calves was almost normal .
the calves were assigned to 
 treatment and corresponding control groups based on the type of scouring ( pathogenic or 
 non - pathogenic ) and clinically healthy ( non - scouring ) status .
the treatment groups included 
 scouring pathogen - positive treated ( ppt ; n=8 ) , scouring pathogen - negative treated ( pnt ; n=8 ) 
 and healthy treated ( ht ; n=6 ) calves .
the corresponding control groups included scouring 
 pathogen - positive control ( ppc ; n=6 ) and pathogen - negative control ( pnc ; n=6 ) calves , and 
 healthy control ( hc ; n=8 ) calves without probiotic treatment were used as the main control 
 group .
all calves received colostrum at birth and were fed a milk replacement twice 
 daily .
treatment of the calves began on the first day of scour occurrence ( day 0 ) .
after a 
 clinical examination and the collection of blood and stool samples , a probiotic ( miyarisan 
 pharmaceutical co. , ltd . , tokyo , japan ) that included l. plantarum strain 
 220 ( 9  10 colony forming units [ cfu]/g ) ,
e. faecium strain 26 
 ( 9  10 cfu / g ) and c. butyricum strain miyari ( 9  
 10 cfu / g ) was administered to each of the calves in the treatment groups once 
 daily at a dose of 3.0 g/100 kg body weight ( bw ) for 5 days .
the probiotic was mixed with a 
 milk replacement and fed to each calf in the morning .
the scouring calves were not treated 
 by any other medicine or electrolyte therapy , although all calves had access to fresh water 
 ad libitum .
blood collection and pbmc isolation : blood samples were collected on days 
 0 and 7 . a blood specimen ( 6 ml )
was collected in a heparinized tube ( bd 
 vacutainer systems , plymouth , u.k . ) and used to isolate pbmcs for flow cytometric analysis . 

blood ( 4 ml ) was also collected in two edta tubes ( bd vacutainer ; becton 
 dickinson , franklin lakes , nj , u.s.a . ) and used for total rna extraction from peripheral 
 leukocytes and for white blood cell ( wbc ) counts .
the 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 the hemogram method 
 with the dif - quick staining protocol .
to purify pbmcs and to lyse red 
 blood cells , 2 ml of the heparinized blood was mixed with 8 
 ml of 0.83% ammonium chloride solution .
after 5 min at room temperature , 
 the samples were centrifuged ( 2,000 rpm , 10 min and 4c ) . the leukocytes were then purified 
 by washing twice in phosphate - buffered saline ( pbs ; ph 7.4 ) followed by centrifugation 
 ( 2,000 rpm , 5 min and 4c ) .
after the final wash , the cell pellet was diluted in 500 
 l of pbs , and the cells were counted .
the cell concentration was then 
 adjusted to 1  10/ml in pbs , and the cells were kept at 4c 
 until they were used for flow cytometry .
flow cytometry : the primary monoclonal antibodies used in the present 
 study are shown in table 1table 1.monoclonal antibodies used for flow cytometryitemtarget moleculespecificitymab cloneisotypesuppliercd3tcr complexpan - t cellsmm1aigg1vmrdcd4cd4 moleculet - helper cellscact183aigg1vmrdcd8cd8cytotoxic t cellsbat82aigg1vmrdcd45rptprpan - leukocytesgc6aigmvmrdwc1unknown t cellsil - a29igg1vmrdcd14lps receptormonocytes , macrophagescam36aigg1vmrdcd21cr2b cellsbaq15aigmvmrdcd282tlr2monocyteshca151figgabd - serotecmab , monoclonal antibody ; tcr , t cell receptor ; ptpr , protein tyrosine phosphatase 
 receptor ; lps , lipopolysaccharide ; cr2 , complement component receptor 2 ; tlr2 , 
 toll - like receptor 2 .. flow cytometry was performed as described by ohtsuka et al . 
 .
pbmcs were labeled with the primary antibody 
 and incubated for 60 min at 4c .
cd3 t cells and each of the 
 cd4 and cd8 t cell subsets were additionally labeled with a 
 monoclonal antibody against cd45r to mark pan - leukocytes .
the labeled cells were then stained with specific 
 purified igg conjugated to a 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 .
after washing twice , the 
 stained cells were diluted in 500 l of pbs .
flow cytometry was performed 
 using a facscan analyzer ( becton dickinson ) equipped with a computer running cell quest 
 software ( becton dickinson ) .
mab , monoclonal antibody ; 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 of 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 u of dnase i per 100 mg of 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 the 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 ) .
the cdna was stored at 80c until it was 
 used for real - time pcr .
real - time pcr : real - time pcr was performed using an iq sybr green supermix 
 kit ( bio - rad ) as described previously .
specific 
 individual cytokine primer sequences and the internal standard gene used ( -actin ) are shown 
 in table 2table 2.primers used for real - time pcrgenegenbank acc .
no.sequence ( 53)annealing temperature ( c)concentration ( nm)-actinay141970f : ggccgagcggaaatcg65750r : gccatctcctgctcgaagtcil-6nm173923f : gtcttcaaacgagtgggtaaag65250r : tgaccagaggagggaatgcil-8nm173925f : cactgtgaaaattcagaaatcattgtta59250r : cttcacaaatacctgcacaaccttctnf-z48808f : cggtggtgggactcgtatg65750r : gctggttgtcttccagcttcaifn-nm174086f : taggcaagtctatgggatttc59250r : gcattcattacatcatcaagtgtable 3a.numbers of peripheral blood mononuclear cells ( pbmcs ) and cd45r 
 /cd45r t cells in the scouring probiotic - treated and scouring control 
 calvesppcpptpncpntday 0day 7day 0day 7day 0day 7day 0day 7pbmcs4,231  4305,658  9523,822  3135,947  554 * 4,427 
4955,126  4254,140  4225,610  393*cd3cd45r1,088  1032,656  565 * 1,064  852,342  282 * 1,185  792,558  401 * 1,375  1272,228  268*cd3cd45r361  281,305  288 * 564  271,394  150 * 678  85968  160 * 552  601,419  131cd4cd45r133  18377  53 * 73  14320  47 * 111  17287  75 * 155 
41205  25*cd4cd45r177  29978  144 * 264  23963  151 * 450  98654  58 * 344  631,049  130cd8cd45r67  15284  41 * 80  43201  22 * 58  7.096  1544  12113  32*cd8cd45r238  41409  73 * 204  16710  142290  51305  60328 
# compared with the values of the corresponding 
 control groups ( p<0.05).table 3b.numbers of peripheral blood mononuclear cells ( pbmcs ) and 
 cd45r / cd45r t cell subpopulations in the healthy 
 probiotic - treated and healthy control calveshchtday 0day 7day 0day 7pbmcs4,500  2385,148  4763,808  2587,218  365*cd3cd45r1,621  1161,725  1981,498  982,003  491*cd3cd45r979  711,114  1521,042  451,815  105cd4cd45r128  34219  51108  21284  22*cd4cd45r482  81439  81501  711,307  96cd8cd45r52  1268  1150  7.5101  14*cd8cd45r409  50502  70421  22693  66*the values ( cells/l ) represent the means  se .
b ) healthy treated calves ( received 3.0 g/100 kg of 
 probiotic for 5 days ; n=6 ) . * compared with day 0 in the same group 
 ( p<0.01 ) .
# compared with the healthy controls on the same day 
 ( p<0.05 ) .. amplification was performed using a mini opticon ( bio - rad ) thermal cycler 
 with the following profile : 1 cycle of 95c for 2 min followed by 45 cycles of 95c for 30 
 sec , and 65 or 59c for 30 sec and 74c for 30 sec followed by 74c for 7 min .
a melting 
 curve analysis was performed from 50 to 95c with readings 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 .
the results are summarized as ct values , where ct 
 is the difference in the threshold cycle for the target and -actin , as an internal 
 control .
* compared with day 0 in the same group 
 ( p<0.01 ) . # compared with the values of the corresponding 
 control groups ( p<0.05 ) .
b ) healthy treated calves ( received 3.0 g/100 kg of 
 probiotic for 5 days ; n=6 ) . * compared with day 0 in the same group 
 ( p<0.01 ) . # compared with the healthy controls on the same day 
 ( p<0.05 ) .
stool analysis : the common pathogens considered essential for calf 
 scouring in the field were examined using stool samples from the calves in the scouring 
 groups . on the first day of scouring , stool samples were obtained from each calf by 
 mechanical stimulation of the anus and preserved in a sterilized tube .
deoxycholate hydrogen 
 sulfide - lactose agar ( elken chemical co. , tokyo , japan ) medium was used for the recognition 
 of salmonella and escherichia coli according to the 
 methods of ishihara et al . .
the 
 cryptosporidium parvum test strip bio k 155 ( bio - x diagnostics , jemelle , 
 belgium ) was used according to the manufacturer s instructions with 0.1 g of stool sample 
 and was evaluated visually . for the detection of coccidia spores ,
for rotavirus , a 
 specific test strip ( sa scientific , san antonio , tx , u.s.a . ) was used following the product 
 use recommendations . among the scouring calves diagnosed as pathogen - positive ( n=16 ) , the 
 pathogens found were rotavirus ( 43% ) , coccidian eimeria bovis and/or 
 eimeria zuernii ( 25% ) , c. parvum ( 19% ) , e. 
 coli ( 13% ) and salmonella ( 0% ) .
those calves with negative stool 
 tests were assigned to the non - pathogenic scouring groups , and clinically healthy calves 
 without scouring were placed in the healthy groups .
reduced occurrence of scouring and an improvement in clinical status 
 were noted in the probiotic - treated calves on day 7 .
statistical analysis : the number of cd - positive or -negative cells was 
 calculated from the percentages of leukocytes gated by flow cytometry and total pbmcs .
graphpad prism ver.5.01 software ( la 
 jolla , ca , u.s.a . ) was used for the statistical calculations and one - way anova , and the 
 kruskal - wallis test was subsequently used to evaluate differences among the groups .
subpopulations of peripheral leukocytes : compared with the values of the 
 hc and ht groups on day 0 , a significantly lower ( p<0.05 ) number of 
 cd3 t cells ; cd4 , cd8 and wc1  t cell 
 subsets ; and cd14 cells were noted in the peripheral blood of scouring ppt and 
 ppc calves . on day 0 , the numbers of cd3 t cells ; the 
 cd4 , cd8 and wc1  t cells subsets ; and 
 cd14 cells were lower ( p<0.05 ) in the ppt and ppc groups 
 than in the other groups .
the number of cd21 cells was similar among the 
 scouring and healthy groups on day 0 ( fig .
1.the numbers of cd3 , cd4 , cd8 , cd14 , cd21 and wc1 cells in the peripheral blood of the 
 scouring ppt ( n=8 ) , ppc ( n=8 ) , pnt ( n=6 ) , pnc ( n=6 ) , ht ( n=6 ) and hc ( n=8 ) groups . 

calves in the treated groups were given 3.0 g/100 kg bw of the probiotic once daily 
 for 5 days , and calves without probiotic treatment served as corresponding controls . 

the first day of 
 scouring occurrence and probiotic treatment was designated as day 0 . ) .
compared to the hc group , cd3 t cells ; the cd4 , 
 cd8 and wc1  t cell subsets ; and cd14 and 
 cd21 cells were increased ( p<0.05 ) in the scouring ppt , 
 pnt and ppc groups on day 7 .
however , the numbers of cd8 and cd21 
 cells were unchanged in the scouring pnc group on day 7 .
the values among the scouring ppt , 
 pnt , ppc and pnc groups were not significantly different on day 7 . moreover , compared with 
 the values on day 0 and those of the hc group , an increased ( p<0.05 ) 
 number of cd3 t cells ; the cd4 , cd8 and wc1 
  t cell subsets ; and cd14 and cd21 cells were noted in the ht 
 group on day 7 .
compared with the value on day 0 , wc1  t cells were increased 
 ( p<0.05 ) in the hc group on day 7 .
likewise , compared with the values 
 on day 0 , pbmcs were increased ( p<0.05 ) in the scouring ppt , pnt and ht 
 groups on day 7 .
a discrete analysis of the data indicated that the numbers of 
 cd45r and cd45r t cells were increased 
 ( p<0.05 ) in the proportion of cd3 
 ( cd3d45r / cd45r ) t cells , cd4 
 ( cd4cd45r / cd45r ) and cd8 
 ( cd8cd45r / cd45r ) t cell subsets in the scouring ppt , 
 pnt , ppc and ht groups on day 7 , compared to the values on day 0 in the same group or the hc 
 group .
however , the values were increased ( p<0.05 ) only in the number of 
 cd3 ( cd3cd45r ) t cells and cd4 
 ( cd4cd45r ) t cell subset in the scouring pnc group on day 7 . the 
 values were not significantly different among the scouring ppt , pnt , ppc and pnc groups 
 ( table 3a and 3b ) .
peripheral 
 cd282 [ toll - like receptor 2 ( tlr2 ) ] monocytes were evaluated only in the 
 scouring ppt , pnt , ht and hc groups .
similar to that of the t cell subpopulations , a 
 significantly lower ( p<0.05 ) number of peripheral tlr2 
 monocytes were noted in the scouring calves on day 0 .
however , the number of 
 tlr2 monocytes was increased ( p<0.05 ) in the scouring ppt , 
 pnt and ht groups on day 7 , compared with the values on day 0 and that of the hc group 
 ( fig . 2fig .
2.number of tlr2 ( cd282 ) cells in the peripheral blood of the scouring ppt ( n=8 ) , pnt 
 ( n=6 ) , ht ( n=6 ) and hc ( n=8 ) groups .
calves in the treated groups were given 3.0 g/100 
 kg bw of the probiotic once daily for 5 days , and calves without probiotic treatment 
 served as corresponding controls .
the values represent the mean  se . * compared to 
 day 0 in the same group .  
the first day of scouring occurrence and probiotic 
 treatment was designated as day 0 . ) .
the numbers of cd3 , cd4 , cd8 , cd14 , cd21 and wc1 cells in the peripheral blood of the 
 scouring ppt ( n=8 ) , ppc ( n=8 ) , pnt ( n=6 ) , pnc ( n=6 ) , ht ( n=6 ) and hc ( n=8 ) groups . 
 calves in the treated groups were given 3.0 g/100 kg bw of the probiotic once daily 
 for 5 days , and calves without probiotic treatment served as corresponding controls . 

the first day of 
 scouring occurrence and probiotic treatment was designated as day 0 .
number of tlr2 ( cd282 ) cells in the peripheral blood of the scouring ppt ( n=8 ) , pnt 
 ( n=6 ) , ht ( n=6 ) and hc ( n=8 ) groups .
calves in the treated groups were given 3.0 g/100 
 kg bw of the probiotic once daily for 5 days , and calves without probiotic treatment 
 served as corresponding controls .
the values represent the mean  se . * compared to 
 day 0 in the same group .   compared to the hc group 
 ( p<0.05 ) .
the first day of scouring occurrence and probiotic 
 treatment was designated as day 0 .
mrna expression of cytokines : compared with the value for the hc calves , 
 the mrna expression levels of interleukin ( il)-6 and interferon - gamma ( ifn- ) were lower 
 ( p<0.05 ) in the scouring ppt , pnt , ppc and pnc groups on day 0 .
the 
 mrna expression level of tumor necrosis factor - alpha ( tnf- ) was lower 
 ( p<0.05 ) in the scouring ppt and ppc groups , but the values were greater 
 in the scouring pnt , pnc , ht and hc groups on day 0 ( fig . 

3.relative mrna expression of il-6 , il-8 , tnf- and ifn- in the peripheral blood of 
 the scouring ppt ( n=8 ) , ppc ( n=8 ) , pnt ( n=6 ) , pnc ( n=6 ) , ht ( n=6 ) and hc ( n=8 ) groups . 

calves in the treated groups were given 3.0 g/100 kg bw of the probiotic once daily 
 for 5 days , and calves without probiotic treatment served as corresponding controls . 

the first day of scouring 
 occurrence and probiotic treatment was designated as day 0 . ) .
il-6 , ifn- and tnf- mrna expression was increased ( p<0.05 ) in 
 the ppt , pnt , ppc , pnc and ht groups on day 7 .
furthermore , compared to day 0 , the tnf- 
 mrna expression level was higher ( p<0.05 ) in the hc group on day 7 .
the 
 mrna expression level of il-8 was similar between the scouring and healthy calves on day 0 , 
 but was elevated ( p<0.05 ) in the scouring ppt and ppc groups on day 7 . 
 compared to the hc group ,
the expression of il-8 mrna was slightly higher in the ht group on 
 day 7 , but the differences were not statistically significant .
relative mrna expression of il-6 , il-8 , tnf- and ifn- in the peripheral blood of 
 the scouring ppt ( n=8 ) , ppc ( n=8 ) , pnt ( n=6 ) , pnc ( n=6 ) , ht ( n=6 ) and hc ( n=8 ) groups . 

calves in the treated groups were given 3.0 g/100 kg bw of the probiotic once daily 
 for 5 days , and calves without probiotic treatment served as corresponding controls . 

the first day of scouring 
 occurrence and probiotic treatment was designated as day 0 .
we recently studied the effects of a probiotic consisting of l. plantarum , 
 e. faecium and c. butyricum on the peripheral leukocyte 
 subpopulations and cytokine mrna expression levels in healthy weaned calves ( in press ) . in 
 the present study , peripheral leukocyte subpopulations and their cytokine mrna expression 
 levels
were evaluated in the field in scouring and healthy pre - weaning calves to clarify the 
 immune - stimulatory effects of the same probiotic agent .
some reports have indicated that a probiotic including lab reduced the rate of git 
 disorders in calves [ 1 , 26 , 35 , 36 ] . increased intestinal lab might stimulate the gut epithelial and 
 associated lymphoid tissues and may activate local immune responses in the intestine .
it was also suggested that a decreased number of 
 peripheral leukocytes , including cd8 and wc1  t cells , might 
 result in a high risk of calf scouring . 

similarly , a lower number of cd4 and cd8 cells and reduced cytokine 
 mrna expression levels were noted in neonates with diarrhea , compared with healthy children 
 [ 9 , 37 ] . in 
 agreement with a previous report by ohtsuka et al . , the numbers of cd4 , cd8 and wc1 
  t cell subsets and of cd14 and tlr2 cells were significantly 
 lower in scouring calves in the present study .
probiotics are known to affect the host 
 immune system in different ways , including inducing greater antibody production and 
 increasing epithelial barrier integrity , the upregulation of cell - mediated immunity , 
 increased tlr signaling , enhanced dendritic cell ( dc)-t cell interactions and heightened t 
 cell associations [ 5 , 18 , 32 ] .
the immune - stimulating properties 
 of a bacillus - based probiotic were suggested by the increase in peripheral 
 blood cd8 cells in scouring calves compared with controls .
furthermore , the number of cd4 cells increased in 
 response to a probiotic consisting of c. butyricum in dairy cows . in a study of calf pbmcs
, the phagocytic activities 
 of leukocytes were increased by a probiotic consisting of l. plantarum 
 .
in addition , selective effects of a probiotic 
 including lactobacillus were reported for t cell activation , 
 cd8 cell activity and cytokine production .
most peripheral t cell subpopulations have cd3 expressed on their surface , 
 which might appear as a signal - transducing unit for the t cell receptor ( tcr ) .
similar to our recent study using the same probiotic 
 agent in healthy weaned calves , the proportion of cd3cd45r t cells 
 was marginally higher in the systemic circulation of pre - weaning scouring and ht calves . 

similar to previous reports , a greater presence of cd45r t cells might 
 facilitate the regular activation of t cell subsets in probiotic - treated calves .
in contrast , bovine wc1  t cells are 
 known to be immune suppressors and are involved in secreting ifn- [ 13 , 28 , 29 ] . wc1  t cells can respond to stimulation via the tcr 
 and through cd3 stimulation , leading to the induction of ifn- expression in these cells 
 .
early production of ifn- by  t cells may 
 have a role in linking innate and adaptive immune responses .
furthermore , bovine  t lymphocyte subsets are activated indirectly through 
 cytokines secreted by t cells , antigen - presenting cells and other accessory cells [ 13 , 28 , 31 ] .
our results show that the number of wc1 
  t cells increased in both the scouring and ht groups and was associated with increased 
 ifn- expression .
in addition , wc1  t cells were also greater in number in the 
 hc group .
this is in accordance with a previous report , which noted that  cell subsets 
 were significantly larger in fetal and maturing calves than in mature animals .
probiotics including lactobacillus and c. 
 butyricum induce the release of il-6 and tnf- , which are involved in 
 nonspecific immune responses .
it was previously 
 reported that pre - weaning calves given a probiotic had increased serum igg and ifn- levels 
 .
furthermore , probiotics including e. 
 faecium and c. butyricum or lactobacillus 
 induced the production of pro- and anti - inammatory cytokines , including il-6 , ifn- and 
 tnf- , in human pbmcs [ 5 , 14 ] . in the present study ,
increased il-6 , inf- and tnf- mrna 
 expression was observed in the scouring treated , ht and hc groups .
in contrast , il-8 mrna 
 expression was unchanged , but it was elevated in the scouring groups . greater expression of 
 il-8 is likely related to neutrophil function and/or inflammatory stimulation in bovines 
 .
therefore , il-8 mrna expression was changed 
 only in scouring calves , but not in healthy calves .
it was reported that tnf- mrna 
 expression was related to age - related changes in intracellular cytokine production . in the present study , a higher tnf- mrna expression 
 level
tlr expression is associated with bacterial immune stimulators , including lab , in the git 
 of calves . in our preliminary study , it was 
 revealed that calves given the probiotic had a stable ruminal ph compared to the controls . 

it was also reported that a significant increase in ruminal ph was correlated with 
 increasing taxonomic units of clostridia in the gastro - intestine of cattle 
 .
however , the bacterial flora was 
 not evaluated in the present study . in bovines ,
the expression of tlr2 was obvious on 
 antigen - presenting cells with a high level expression on peripheral monocytes and 
 gastrointestinal macrophages [ 19 , 21 , 25 ] .
moreover , 
 bovine tlr2 cell surface molecules on monocyte - derived dcs and intestinal macrophages are 
 most responsible for the recognition of gram - positive bacteria [ 3 , 6 , 10 ] . in human pbmcs
, monocytes presenting tlrs played an important role 
 in lactobacillus - induced immunological responses .
mechanisms indicating a direct role for tlr2 include bacterial 
 reorganization by epithelial macrophages in calves .
however , cd14 cells are involved with the tlr4 and md2 complex , 
 which is essential for the response to most gram - negative bacteria [ 19 , 25 ] . among probiotics , 

lactobacillus strains have a rigid cell wall that is resistant to 
 intracellular digestion and can stimulate intestinal macrophages presenting tlr2 to secrete 
 a large quantity of cytokines . in the present 
 study ,
the numbers of cd14 and tlr2 cells were increased in the 
 scouring and ht calves compared to the controls . a b - cell receptor complex including cd21 is 
 present on mature b cells and ileal peyer s patch - derived b lymphocytes in young calves 
 .
the stimulation of cd21 cells is 
 related to intestinal immune stimuli , and probiotics are known to trigger naive plasma cells 
 to become iga - producing cd21 b cells [ 12 , 
 24 , 38 ] . in 
 our study , the number of cd21 cells was increased in the scouring and healthy 
 probiotic - treated calves on day 7 .
an interaction between probiotics and the host immune system leads to the interface of 
 intestinal immune cells , such as cd14 and tlr cell macrophages or 
 dcs and to the release of cytokine stimulators by t and b cells [ 6 , 8 , 14 ] .
the activation and balanced response of t cells is an important 
 component of the recovery from git disease in cattle [ 25 , 38 ] .
probiotics contribute to 
 homeostasis of the intestinal bacterial flora and result in scouring recovery in young 
 calves [ 26 , 35 , 36 ] . in the present study ,
a probiotic was administed to pre - weaning scouring calves for 5 days , 
 and stool samples were examined to discover the underlying cause of scouring .
the scouring 
 ppt and ppc or pnt and pnc groups revealed a greater number of peripheral leukocytes and 
 higher cytokine mrna expression levels on day 7 , although the immune marker values were 
 slightly higher in the ppt and ppc groups compared with pnt and pnc groups .
higher immune 
 marker values on day 7 in the both pathogen - positive and pathogen - negative scouring groups 
 ( ppt and pnt ) might be induced by probiotic administration and scouring causers . 
 furthermore , the increased numbers of circulating immune cells in ht group indicated the 
 immune - stimulatory effects of the probiotic , as suggested by previous studies [ 7 , 20 ] .
on the 
 other hand , the scouring calves given no probiotic ( ppc and pnc groups ) exhibited greater 
 expression of immune markers on day 7 , which might be associated with the usual reaction of 
 immune system through the git disorders .
the 
 recovery of scouring in the calves given probiotic was in agreement with that in previous 
 reports [ 1 , 12 , 
 19 , 26 ] . 
 from these findings , probiotic administration to the scouring calves might be effective in 
 both pathogen - positive and pathogen - negative cases . in this study ,
however , it was previously suggested that the serum igg concentration was 
 increased by probiotic administration in pre - weaning calves . in conclusion
, the administration of a bacteria - based probiotic resulted in increased 
 numbers of cd3 t cells ; cd4 , cd8 and wc1  
 t cells ; and cd14 , cd21 and tlr2 cells in scouring ppt , 
 pnt and ht calves , compared with healthy controls .
moreover , higher il-6 , inf- and tnf- 
 mrna expression levels were noted in peripheral leukocytes , and the probiotic - treated calves 
 recovered from scouring .
these findings demonstrate that the administration of a probiotic 
 elicits immunological changes in the systemic circulation of scouring and healthy calves . 

considering the effects of probiotics on immune markers in healthy calves , the repeated 
 treatment of healthy pre - weaning calves with a 5-day probiotic regimen could elevate the 
 levels of reduced immune markers , thereby reducing the incidence of scouring in young 
 calves .
 abstractsubpopulations of peripheral leukocytes and cytokine mrna expression levels 
 were evaluated in scouring and healthy holstein calves ( age 10  5 days ; n=42 ) treated 
 with a probiotic consisting of lactobacillus plantarum , 
 enterococcus faecium and clostridium butyricum . 
 the 
 calves were assigned to the scouring or healthy group and then subdivided into 
 pathogen - positive treated ( n=8 ) , pathogen - positive control ( n=8 ) , pathogen - negative 
 treated ( n=6 ) , pathogen - negative control ( n=6 ) , healthy treated ( n=6 ) and healthy control 
 ( n=8 ) groups . 
 a single dose of the probiotic ( 3.0 g/100 kg body weight ) was given to each 
 calf in the treatment groups for 5 days . 
 blood samples were collected on the first day of 
 scour occurrence ( day 0 ) and on day 7 . in the scouring calves , smaller peripheral 
 leukocyte subpopulations and cytokine mrna expression levels were noted on day 0 . 
 the 
 numbers of cd3 + t cells ; cd4 + , cd8 + and wc1 + 
  t cell subsets ; and cd14 + , cd21 + and cd282 + ( tlr2 ) 
 cells were significantly increased in the scouring and healthy treated calves on day 7 . 
 
 furthermore , interleukin-6 , tumor necrosis factor - alpha and interferon - gamma mrna 
 expression was elevated in the peripheral leukocytes of the scouring and healthy treated 
 calves on day 7 . 
 the scouring calves given the probiotic recovered on day 7 . 
 a 
 significantly smaller number of peripheral leukocytes and lower cytokine mrna expression 
 level might be induced by scouring in calves . 
 repeated probiotic administration might 
 stimulate cellular immunity and encourage recovery from scouring in pre - weaning holstein 
 calves . 
MATERIALS AND METHODS
RESULTS
DISCUSSION