clinical symptoms in diseased calves : case 1 : in may 2010 , a holstein calf 
 ( female , 84 days old ) showed anorexia , pyrexia and diarrhea in a depositary raising farm in 
 yamaguchi prefecture . despite treatment with antibiotics ( kanamycin and tylosin ) , 
 sulfamonomethoxine and antipyretic , the calf died 20 days after the onset of diarrhea . case 
 2 : in july 2012 , a japanese black calf ( female , 246 days old ) on another farm in yamaguchi 
 prefecture repeated diarrhea despite treatment with antibiotics ( kanamycin ) and a probiotic 
 product after the birth , and then , feces were collected during the acute phase for 
 diagnosis .
isolation of microorganisms : tissue specimens obtained from major organs 
 including cerebrum , jejunum , ileum , cecum , colon and ideal contents of the dead calf ( case 
 1 ) and feces of the second calf ( case 2 ) were minced and homogenized in serum - free eagle s 
 minimum essential medium with kanamycin ( mem ) ( nissui pharmaceutical co. , tokyo , japan ) . 

after centrifugation of the 10% homogenate , the supernatant was filtered through a 450 nm 
 membrane ( merck millipore ltd . , carrigtwohill , ireland ) , and 0.15 ml of the 
 supernatants were inoculated onto hamster lung ( hmlu-1 ) cells , mardin - darby bovine kidney 
 ( mdbk ) cells , bovine testicular ( bt ) cells , human rectal adenocarcinoma ( hrt-18 ) cells and 
 vero cells in 24-well plates .
after adsorption for 60 min at 37c , the cells were washed 
 with mem , and then , 0.5 ml of mem containing 0.1% bovine serum albumin 
 ( bovogen biologicals , williams avenue , australia ) was added to each well .
the cells were 
 incubated at 37c , and cytopathic effects ( cpe ) were observed .
after incubation for 10 days , 
 cells were frozen and thawed once and then centrifuged .
subsequent passages were carried out 
 at least twice in the same manner with 0.15 ml of the supernatant .
dhl agar , columbia agar with 5% sheep blood and gam agar 
 with 5% yolk and 0.1% cysteine were used for bacterial isolation .
the gene of 
 clostridium perfringens toxin isolated from small - intestinal contents was 
 identified by pcr .
identification of isolates by pcr , sequence and phylogenetic analysis of c. pecorum 
 omp1 gene : c. pecorum - infected cells and the supernatants from 
 organs described above were used for dna extraction .
total dna was extracted using dneasy 
 blood & tissue kit ( qiagen , hiden , germany ) .
pcr was carried out with takara ex taq hot 
 start version ( takara bio inc .
primer pairs targeting fragments of 
 genus - specific and species - specific chlamydia omp1 genes were used for pcr 
 as described by kaltenbck et al . .
pcr products were electrophoresed on 2.0% agarose gel and visualized using 
 ethidium bromide staining .
pcr products were purified using minielute pcr purification kit 
 ( qiagen , germantown , md , u.s.a . ) and directly sequenced using a bigdye terminator cycle 
 sequencing kit v3.1 ( applied biosystems , austin , tx , u.s.a . ) .
phylogenetic trees were 
 constructed using neighbor - joining methods , and 
 the reliability of the branch was evaluated by bootstrapping with 1,000 replicates .
antigenic analysis of c. pecorum isolates : the antigenicity of isolates 
 were compared by immunoblot analysis using rabbit antisera   to bo / yokohama , bo / maeda   and ov / ipa 
 strains of c. pecorum .
antisera to bo / maeda and ov / ipa strains were 
 prepared as described by fukushi and hirai .
hmlu-1 
 cells were infected with c. pecorum 2258 , bo / yokohama , bo / maeda and ov / ipa 
 strains and c. psittaci cal10 
 strain and then incubated at 37c in 5% co2 until cpe was observed .
the cells 
 were recovered from dishes with 0.02% edta in phosphate - buffered saline ( pbs ) .
after 
 centrifugation at 200  g for 5 min at 4c , the supernatant was removed , 
 and the cells were resuspended in pbs .
cells were then mixed with an equal volume of 2  
 concentrated sample buffer consisting of 6.25 mm tris - hcl ph 6.8 , 2% sodium dodecyl sulfate 
 ( sds ) , 20% glycerol and 0.001% bromophenol blue .
samples were boiled for 3 min , placed on 
 ice for 3 min and centrifuged at 13,000  g for 3 
 min at room temperature .
cell lysates were electrophoresed using 12% sds - page 
 and transferred to a pvdf membrane ( immobilon - p ; millipore , billerica , ma , u.s.a . ) .
after 
 blocking with tris - buffered saline ( tbs ) containing 3% gelatin ( eia grade reagent gelatin ; 
 bio - rad , ca , u.s.a . ) for 45 min at 37c , the membrane was washed three times with tbs 
 containing 0.05% tween 20 ( t - tbs ) .
after incubation with diluted rabbit antisera for 45 min 
 at 37c , the membrane was washed 3 times with t - tbs . then , the membrane was reacted with 
 peroxidase - conjugated purified recombinant protein a / g ( thermo fischer scientific , rockford , 
 il , u.s.a . ) for 45 min at 37c .
after washing the membrane with t - tbs and tbs three times 
 each , specific bands were visualized using 3,3-diaminobenzidine tetrahydrochloride ( dab ; 
 wako pure chemical industries , ltd . , osaka , japan ) .
each dilution was mixed with an 
 equal volume of 200 tcid50/0.05 ml of c. 
 pecorum strains and incubated at 37c for 1
then , 0.1 ml 
 ( approximately 6  10 cells ) of hmlu-1 cells in mem containing 10% fetal bovine 
 serum was added to each well .
after incubation at 37c for 7 days , antibody titers were 
 expressed as the reciprocal of the highest dilution of serum which inhibited cpe 
 completely .
histological and immunohistochemical analyses : tissue specimens obtained 
 from major organs , intestines and central nervous system of the dead calf ( case 1 ) were 
 fixed in 10% neutral buffered formalin and embedded in paraffin wax .
paraffin - embedded 
 tissues were cut at 2 m and stained with hematoxylin - eosin .
after deparaffinization and rehydration of the paraffin 
 sections , endogenous peroxidase activity was blocked by incubation in 3% 
 h2o2 for 10 min .
sections were incubated with 10% normal goat serum 
 for 30 min and then with a rabbit anti - c .
sections were 
 rinsed three times in pbs containing 0.2% tween 20 for 10 min and then incubated with 
 histofine simple stain max - po ( r ) ( nichirei biosciences , inc . , tokyo , japan ) for 30 min .
the 
 sections were rinsed three times in pbs containing 0.2% tween 20 for 10 min .
the sections 
 were incubated with 3,3-diaminobenzidine tetrahydrochloride under the microscope to control 
 color development , washed in tap water for 10 min and rinsed with distilled water .
isolation of microorganisms and pcr for virus detection : characteristic 
 cpe appeared in hmlu-1 cells inoculated with jejunum homogenate from case 1 ( 2258 strain ) 
 at the first passage ( fig .
1.characteristic cpe observed in hmlu-1 cells inoculated with homogenized jejunum 
 supernatant from case 1 . ) .
cpe was observed seven days after inoculation . on the other hand , at the second 
 passage , cpe similar to that of 2258 strain appeared in hmlu-1 cells inoculated with calf 
 feces from case 2 ( 24100 strain ) .
gimenez staining of hmlu-1 cells infected with these 
 strains revealed many small purplish - red particles . c.
in addition , c. pecorum - specific 
 genes were detected from jejunum , ileum , cecum and colon of case 1 .
clostridium 
 perfringens was isolated from jejunal and ileal contents of case 1 at 
 approximately 10 cfu / g .
the gene of -toxins was shown in these isolates by pcr , 
 and isolates were identified as type a ( data not shown ) .
characteristic cpe observed in hmlu-1 cells inoculated with homogenized jejunum 
 supernatant from case 1 .
no other viruses were isolated from specimens , and pcr was also negative for all specimens 
 from cases 1 and 2 when tested for bovine viral diarrhea virus , bovine coronavirus , 
 rotavirus [ 3 , 10 , 25 ] , bovine torovirus , bovine enterovirus , paramyxoviruses , bovine 
 adenovirus   and bovine herpesvirus 4 .
identification of isolates by pcr , sequence and phylogenetic analysis : two 
 isolates ( 2258 and 24100 ) were pcr positive when genus - specific and species - specific 
 omp1 genes of c. pecorum were targeted .
nucleotide 
 sequences of genus - specific pcr products were determined and compared with those of 
 published c. pecorum omp1 genes .
omp1 gene partial 
 sequence of the 2 isolates was 100% identical and showed the highest identity ( 99.8% ) to the 
 bo / yokohama strain isolated from cattle with enteritis in japan .
nucleotide similarities of 
 the omp1 gene between the isolates and other published c. 
 pecorum isolates were 89.2% , 85.2% and 82.0% for bo / maeda strain which was 
 isolated from cattle with pneumonia in japan , ov / ipa strain which was isolated from sheep 
 with polyarthritis in united states , and 66p130 strain which was isolated from cattle with 
 enteritis in united states , respectively ( fig . 

2.phylogenetic tree of c. pecorum strains based on the nucleotide 
 sequence of the omp1 gene ( 534 nucleotides ) .
genbank accession number are 22 - 58 
 ( lc021417 ) , 24 - 100 ( lc021418 ) , bo / yokohama ( lc021422 ) , bo / maeda ( lc021419 ) , ov / ipa 
 ( lc021421 ) , l71 ( af269280 ) , lw613 ( aj440240 ) , sbe ( eu684916 ) , ab10 ( eu684917 ) , m14 
 ( eu684920 ) , 824 ( eu684922 ) , be53 ( eu684923 ) , ib1 ( eu684924 ) , ib2 ( eu684925 ) , ib3 
 ( eu684926 ) , ib4 ( eu684927 ) , ib5 ( eu684928 ) , w73 ( eu684929 ) , r69 ( eu684930 ) , ic3 
 ( eu684932 ) , ic4 ( eu684933 ) , 1710s ( gq228167 ) , 1920brz ( gq228168 ) , 66p130 ( gq228180 ) , 
 1708 ( gq228194 ) and mc marsbar ugt ( hq457473 ) .
the nucleotide sequence of c. 
 psittaci cpx0308 strain ( ab284064 ) was used as an outgroup to root the 
 tree .
the percentage bootstrap values calculated from 1,000 replications are indicated 
 above the internal nodes . ,
table 1table 1.nucleotide sequence identities ( % ) of omp1 genes among 
 c. pecorum strainsstrain225824100bo / yokohamabo / maedaov / ipa66p1302258 - 10099.889.285.282.024100 - 99.889.285.282.0bo / yokohama-89.085.081.8bo / maeda-84.283.0ov / ipa-80.266p130- ) .
phylogenetic tree of c. pecorum strains based on the nucleotide 
 sequence of the omp1 gene ( 534 nucleotides ) .
genbank accession number are 22 - 58 
 ( lc021417 ) , 24 - 100 ( lc021418 ) , bo / yokohama ( lc021422 ) , bo / maeda ( lc021419 ) , ov / ipa 
 ( lc021421 ) , l71 ( af269280 ) , lw613 ( aj440240 ) , sbe ( eu684916 ) , ab10 ( eu684917 ) , m14 
 ( eu684920 ) , 824 ( eu684922 ) , be53 ( eu684923 ) , ib1 ( eu684924 ) , ib2 ( eu684925 ) , ib3 
 ( eu684926 ) , ib4 ( eu684927 ) , ib5 ( eu684928 ) , w73 ( eu684929 ) , r69 ( eu684930 ) , ic3 
 ( eu684932 ) , ic4 ( eu684933 ) , 1710s ( gq228167 ) , 1920brz ( gq228168 ) , 66p130 ( gq228180 ) , 
 1708 ( gq228194 ) and mc marsbar ugt ( hq457473 ) .
the nucleotide sequence of c. 
 psittaci cpx0308 strain ( ab284064 ) was used as an outgroup to root the 
 tree .
the percentage bootstrap values calculated from 1,000 replications are indicated 
 above the internal nodes .
antigenic characterization of c. pecorum isolates : the 38 to 40 kda major 
 outer membrane protein   of 2258 strain strongly 
 reacted with rabbit antiserum raised against the bo / yokohama strain . on the other hand ,
no 
 specific reaction was detected by immunoblot analysis with rabbit antisera to bo / maeda or 
 ov / ipa strains ( fig . 3fig .
antiserum against bo / yokohama ( a ) , bo / maeda ( b ) and ov / ipa ( c ) 
 strains was used for each figure .
lane 1 : mock - infected hmlu-1 cells ; lane 2 , 2258 
 strain - infected hmlu-1 cells ; lane 3 , bo / maeda strain - infected hmlu-1 cells ; lane 4 , 
 bo / yokohama strain - infected hmlu-1 cells ; lane 5 , ov / ipa strain - infected hmlu-1 cells ; 
 lane 6 , c. psittaci cal10 strain - infected hmlu-1 cells . arrows 
 indicate a specific band of major outer membrane protein ( 3840 kda ) . ) .
antigenicity between isolates ( 2258 and 24100 ) and the 3 strains ( bo / yokohama , 
 bo / maeda and ov / ipa ) was compared using cross - neutralization tests with rabbit antisera
. 
 isolates 2258 and 24100 were antigenically similar to the bo / yokohama strain , but were a 
 little different from the bo / maeda and ov / ipa strains ( table 2table 2.cross-reactivity among c. pecorum strains determined by 
 neutralization teststrainantibody titer to antisera againstbo / yokohamabo / maedaov / ipa22588<2<2241004<2<2bo / yokohama164<2bo / maeda48<2ov / ipa<2<24a ) the reciprocal of the highest dilution of serum which inhibited cpe 
 completely . ) .
antiserum against bo / yokohama ( a ) , bo / maeda ( b ) and ov / ipa ( c ) 
 strains was used for each figure .
lane 1 : mock - infected hmlu-1 cells ; lane 2 , 2258 
 strain - infected hmlu-1 cells ; lane 3 , bo / maeda strain - infected hmlu-1 cells ; lane 4 , 
 bo / yokohama strain - infected hmlu-1 cells ; lane 5 , ov / ipa strain - infected hmlu-1 cells ; 
 lane 6 , c. psittaci cal10 strain - infected hmlu-1 cells .
arrows 
 indicate a specific band of major outer membrane protein ( 3840 kda ) .
a ) the reciprocal of the highest dilution of serum which inhibited cpe 
 completely .
neutralization test for paired sera of case 2 : a significant increase in 
 antibody against 24100 strain was confirmed by neutralizing tests using paired sera . 
 neutralizing titers in sera collected during the acute phase of infection were less than 
 1:2 , but increased to 1:4 during the convalescent phase .
histopathology and immunohistochemical staining for c. pecorum antigens in tissues 
 from case 1 : systemic hyperemia and congestion together with necrosis of villi 
 with short or long rod - shaped bacteria in the upper part of the small intestine were 
 observed in tissue sections .
hemorrhagic ileitis with edematous and granulomatous changes 
 was observed in the intestinal crypts of the ileum .
positive reactions were observed in the jejunal villi by immunohistochemical 
 staining with rabbit anti - c .
in this study , c. pecorum was isolated from specimens obtained from 2 
 calves with diarrhea . in case 1 ,
the bacterium was isolated from jejunum , and c. 
 pecorum specific omp1 genes were detected from several locations 
 in the intestines . additionally , c. pecorum antigens were observed in the 
 jejunal villi by immunohistochemical staining .
therefore , we 
 speculated that c. pecorum might exacerbate the disease caused by 
 clostridium perfringens . in these 2 cases , it is possible that c. pecorum was either the primary 
 pathogen or an exacerbating factor causing diarrhea . on the other hand
recently , poudel et al .   reported that asymptomatic endemic c. pecorum infections 
 reduce growth rates in calves by up to 48% .
they considered that the mechanism of growth 
 suppression by subclinical chlamydial infection was malabsorption of nutrients due to a 
 local inflammatory response to intestinal mucosal infection .
additionally , despite the 
 absence of clinical signs , chlamydial infection was associated with reduced serum iron 
 concentrations and lower hematocrit values , and infected calves were leukopenic .
mohamad and rodolakis   reported that the persistence of c. pecorum strains 
 in the intestines and vaginal mucus of ruminants could cause long - term sub - clinical 
 infection which may affect the animal s health .
this may explain the poor weight gain 
 observed in case 2 after recovery from diarrhea .
although the standard method for detecting antibodies to chlamydiaceae 
 spp . in animals is still the complement fixation test , our study showed that a neutralization test was also a useful method for 
 diagnosis of c. pecorum infections .
in addition , in the dead calf , it was 
 confirmed that immunohistochemistry for c. pecorum antigens was also 
 useful .
genetic and antigenic analysis showed that 2258 and 24100 strains were more closely 
 related to bo / yokohama strain isolated from cattle with enteritis than to bo / maeda strain 
 isolated from cattle with pneumonia and ov / ipa strain isolated from sheep with 
 polyarthritis .
bo / yokohama - like c. pecorum strains might cause enteritis 
 more than other serotypes .
however , the 2 isolates showed higher sequence identities to 
 bo / maeda and ov / ipa strains than to 66p130 strain isolated from cattle with enteritis in 
 united states .
thus , sequence identities of c. pecorum omp1 gene might vary 
 according to their geographical background .
the isolation of similar strains from different locations in separate years suggests that 
 c. pecorum might be spreading among the cattle population in yamaguchi 
 prefecture .
an epidemiological study of c. pecorum is being conducted 
 currently to clarify the seroprevalence and relationship with disease . in conclusion
, this study showed that c. pecorum isolates similar to 
 bo / yokohama might be endemic in yamaguchi prefecture and cause enteric diseases in 
 cattle .
 chlamydia pecorum ( designated 2258 ) was isolated in 2010 in 
 hmlu-1 cells from the jejunum of a calf which died of necrotizing enterocolitis in 
 yamaguchi prefecture , japan . 
 immunohistochemical staining identified c. 
 pecorum positive reactions in the jejunal villi . 
 c. pecorum , 
 designated 24100 , was isolated from the feces of a calf with diarrhea in another farm in 
 yamaguchi prefecture in 2012 . a significant increase in neutralizing antibody titers 
 against c. pecorum 
 was confirmed in paired sera . 
 nucleotide sequence 
 identities of omp1 genes of the 2 isolates were 100% . 
 the isolates were 
 genetically and antigenically more closely related to c. pecorum 
 bo / yokohama strain isolated from cattle with enteritis in japan than to the other 
 prototype strains , bo / maeda isolated from cattle with pneumonia and ov / ipa isolated from 
 sheep with polyarthritis . 
 these results indicate that c. pecorum strains 
 similar to 2258 and 24100 might be endemic in yamaguchi prefecture and cause enteric 
 disease in cattle . 
MATERIALS AND METHODS
RESULTS
DISCUSSION