ethics : the animal care and use committee of the national institute of 
 animal health ( niah ) approved all animal procedures prior to initiation of this study 
 ( authorization numbers : 12 - 027 , 12 - 056 , 12 - 081 , 13 - 024 , 13 - 054 and 14 - 009 ) .
all experimental 
 infections were performed in cubicles whose sizes have approximately 14 m in a 
 high - containment facility at the niah .
experimental infections in non - vaccinated animals : full details of 
 experimental infections ( i ) , ( ii ) and ( iii ) have already been published [ 11 , 14 ] .
brief 
 details of the experimental infections that provided serum samples for this study are as 
 follows : ( i ) two 6-month - old holstein cattle housed in separate cubicles were inoculated 
 with 1 ml of 10 50% tissue culture infectious dose 
 ( tcid50 ) of the fmdv o / jpn/2010 - 1/14c 
 by an intradermal route . at 1 day post - infection ( dpi ) , two additional 6-month - old holstein 
 cattle were housed with the infected cattle .
( ii ) two 4-month - old 
 japanese saanen goats were inoculated with 1 ml of 10 
 tcid50 of the o / jpn/2010 - 1/14c by an intradermal route . at 1 dpi , 2 additional 
 4-month - old japanese saanen goats were housed with the infected goats .
( iii ) thirteen 2-month - old pigs were inoculated with 1 ml of 10 
 or 10 tcid50 of the fmdv o / jpn/2010 - 1/14c by intranasal or intraoral 
 route .
experimental infections in vaccinated animals : ( iv ) seven 3-month - old 
 holstein cattle were administered intramuscularly an inactivated fmdv vaccine ( six 50% 
 protection dose ( pd50 ) , serotype o , o manisa strain , aftpor , merial , lyon , 
 france ) . at 3 or 30 days post - vaccination ( dpv ) , the vaccinated cattle were inoculated with 
 1 ml of 10 tcid50 of the fmdv o / jpn/2010 - 1/14c by an 
 intradermal route .
( v ) six 2-month - old pigs were administered intramuscularly the fmdv vaccine ( 6 
 pd50 ) . at 3 or 30
dpv , the vaccinated pigs were inoculated with 1 
 ml of 10 tcid50 of the fmdv o / jpn/2010 - 1/14c by an 
 intraoral route .
collection of clinical samples from the animals : the clinical samples were 
 collected routinely as shown in tables 1 
 table 1.detection of viral genes from clinical samples by rt - pcr assay and of 
 antibodies by lpbe and the priocheck kit in infected cattle without 
 vaccination , 2 
 table 2.detection of viral genes from clinical samples by rt - pcr assay and of antibodies 
 by lpbe and the priocheck kit in infected goats without vaccination , 3table 3.detection of antibodies by lpbe and the priocheck kit in infected pigs by the 
 intranasal and intraoral routes without vaccination , 4table 4.detection of viral genes from clinical samples by rt - pcr assay and of antibodies 
 by lpbe and the priocheck kit in infected cattle with vaccination , 5table 5.detection of viral genes from clinical samples by rt - pcr assay and of antibodies 
 by lpbe and the priocheck kit in infected pigs with vaccination .
sera were collected from cervical veins using a vacuum blood 
 collection tube ( venoject ii , terumo corporation , tokyo , japan ) .
saliva was collected from 
 oral cavities using a roll - shaped synthetic saliva collector ( salivette , sarstedt kk , tokyo , 
 japan ) and forceps .
nasal swabs were collected from nasal cavities using a cotton swab 
 ( men - tip , jcb industry limited , tokyo , japan ) .
rna extraction and rt - pcr : viral rnas were extracted from the clinical 
 samples using the high pure viral rna kit ( roche diagnostics , basel , switzerland ) .
the 
 fmdv - specific genes were detected by an rt - pcr assay using primers fm8 and fm9 , which can 
 amplify a portion of a 3d region of an fmdv genome .
lpbe and nsp - elisa : an lpbe ( institute for animal health , surrey , u.k . ) 
 was performed for detection of antibodies to structural proteins ( sps ) of fmdv according to 
 manufacturer s instructions .
the priocheck fmdv ns ( thermo fisher scientific , waltham , ma , u.s.a . )   was performed for detection of antibodies to the non - structural 
 protein 3abc of fmdv according to the manufacturer s instructions .
infected cattle without vaccination : in cattle 1 and 2 , which were 
 inoculated with the fmdv , clinical signs , such as vesicular development on the tongues , 
 inside lips , snouts and feet , excess salivation , and lameness , were initially found between 
 1 and 6 dpi .
viral genes were detected from the clinical samples between 1 and 9 dpi ( table 1 ) .
the days when the antibodies were first detected in the priocheck kit 
 were delayed for 2 days from those in the lpbe . in cattle 3 and 4 , which were housed with the infected cattle , clinical signs , such as 
 vesicular development on the tongues , inside lips , snouts and feet , and excess salivation , 
 were initially found between 4 and 6 days post - contact ( dpc ) .
viral genes were detected from 
 the clinical samples between 2 and 8 dpc ( table 
 1 ) .
the days when 
 the antibodies were first detected in the priocheck kit were delayed for 3 days from those 
 in the lpbe .
infected goats without vaccination : in goats 1 and 2 , which were 
 inoculated with the fmdv , clinical signs , such as vesicular development on the feet , were 
 initially found between 3 and 4 dpi .
viral genes were detected from the clinical samples 
 between 1 and 8 dpi ( table 2 ) .
in the priocheck kit , the antibodies were detected from 5 and 4 dpi , 
 respectively .
the day when the antibody was first detected in the priocheck kit was 1 day 
 after that by the lpbe in goat 1 ; in goat 2 , antibodies were detected by both tests on the 
 same day . in goats 3 and 4 , which were housed with the infected goats , clinical signs , such as 
 vesicular development on the tongue , inside lip , snout and feet , and excess salivation ,
viral genes were detected from the clinical samples 
 between 3 and 11 dpc ( table 
 2 ) .
the antibodies were first detected 1 day 
 later by the priocheck kit than by the lpbe .
infected pigs by the intranasal route without vaccination : in pigs 124 to 
 126 , which were inoculated with 10 tcid50 of the fmdv , clinical 
 signs , such as vesicular development on the inside lips , snouts , teat and feet , excess 
 salivation , lameness , depression and reduced appetite , were initially found between 3 and 9 
 dpi .
the days when viral genes were detected in 
 pigs 124 to 126 were as described previously .
as 
 previously reported , antibodies were detected 
 from 8 , 8 and 7 dpi in the lpbe , respectively ( table 
 3 ) .
the days when the antibodies 
 were first detected in the priocheck kit were delayed for 1 , 1 and 6 days from those in the 
 lpbe , respectively . in pigs 121 to 123 , viral genes and lpbe antibodies were not detected during the 
 experimental period as described previously . in 
 the priocheck kit , antibodies were not also detected in this study . infected pigs by the intraoral route without vaccination : in pigs 131 to 
 133 , which were inoculated with 10 tcid50 of the fmdv , clinical 
 signs , such as vesicular development on the tongue , inside lips , snouts and feet , excess 
 salivation , lameness and depression , were initially found between 7 and 10 dpi .
the days when viral genes were detected in pigs 131 
 to 133 were as described previously . as 
 previously reported , antibodies were detected 
 from 13 , 10 and 12 dpi in the lpbe , respectively ( table 
 3 ) .
the days when the antibodies were first 
 detected in the priocheck kit were delayed for 1 , 1 and 2 days from those in the lpbe , 
 respectively . in pigs 134 to 137 , which were inoculated with 10 tcid50 of the 
 fmdv , clinical signs , such as vesicular development on the snout and feet , excess 
 salivation , lameness , depression and reduced appetite , were initially found between 1 and 6 
 dpi .
the days when viral genes were detected in 
 pigs 134 to 137 were as described previously .
as 
 previously reported , antibodies were detected 
 from 6 , 6 , 7 and 7 dpi in the lpbe , respectively ( table 
 3 ) .
the antibodies 
 were detected from 7 , 7 , 9 and 13 dpi , respectively .
the days when the antibodies were first 
 detected in the priocheck kit were delayed for 1 , 1 , 2 and 6 days from those in the lpbe , 
 respectively . infected cattle with vaccination : in cattle 0 , 1 and 6 , which were 
 vaccinated at 30 days before virus infection ( dbv ) , any clinical signs , such as vesicular 
 development , excess salivation , lameness , depression and reduced appetite , were not found , 
 except for development of lesions on sites of the virus infection .
viral genes were detected 
 from the clinical samples between 1 and 7 dpi ; however , they were not detected in cattle 1 
 during the experimental period ( ) . antibodies were detected from 23 dbv in the lpbe .
using the priocheck kit , the antibodies were detected from 6 , 3 and 7 
 dpi , respectively . in cattle 3 , 7 , 2 and 9 , which were vaccinated at 3 dbv , clinical signs , such as vesicular 
 development on the tongues , inside lips , snouts and feet ,
viral genes were detected from the clinical samples between 1 and 7 dpi ( table 4 ) .
antibodies were detected from 3 , 3 , 2 and 
 2 dpi in the lpbe , respectively .
the antibodies were detected from 5 , 5 , 6 and 5 dpi , respectively .
however , in cattle 
 2 , no antibody was detected at 14 dpi in the priocheck kit .
infected pigs with vaccination : in pigs 1 to 3 , which were vaccinated at 
 30 dbv , any clinical signs , such as vesicular development , excess salivation , lameness , 
 depression and reduced appetite , were not found .
viral genes were detected from the clinical 
 samples between 1 and 7 dpi ( table 5 ) .
an antibody was first detected at 3 dbv . in pig 2 , although 
 an antibody was first detected at 20 dbv , there were several days when antibodies were not 
 detected until 1 dbv . in pig 3
, antibodies were detected at 10 and 17 dbv ; however , 
 antibodies were not detected from 3 dbv to 4 dpi . in the priocheck kit
, antibody responses 
 of the pigs also varied . in pigs 1 and 2 ,
antibodies were not detected during the 
 experimental period in this study , except for 7 dpi in pig 2 .
an antibody was first detected 
 at 18 dpi in pig 3 . in pigs 5 and 6 , which were vaccinated at 3 dbv , clinical signs , such as vesicular 
 development on the tongues , snout and feet ,
however , any clinical signs , such as vesicular development , excess salivation , lameness , 
 depression and reduced appetite , were not found in pig 4 .
viral genes were detected from the 
 clinical samples between 1 and 18 dpi ( table 5 ) . 

antibodies were detected from 4 , 11 and 6 dpi in the lpbe , respectively .
in the priocheck , antibodies were detected from 11 and 7 dpi in 
 pigs 5 and 6 , respectively ; however , the antibodies were detected intermittently between 11 
 and 34 dpi in pig 4 .
the tests are in principle divided into 2 kinds of 
 tests ; those detecting antibodies to sps and to nsps of fmdv , respectively .
there are 7 
 serotypes in fmdv : a , o , c , sat1 , sat2 , sat3 and asia1 .
an appropriate antigen 
 needs to be used for measuring antibodies to a seeking serotype .
in contrast to tests for 
 sps , tests for nsps are independent of serotypes . tests for nsps therefore may be more 
 convenient in countries where several serotypes of fmdvs are prevalent . according to the 
 terrestrial animal health code
, tests for both 
 sps and nsps can be used for serological surveillance in areas where animals are not 
 vaccinated . in practice ,
tests for nsps have been frequently used for serological 
 surveillance of cattle in south american countries [ 1 , 
 2 ] .
however , the results in this study showed that 
 the first detection of antibodies by the priocheck kit was delayed compared to that by the 
 lpbe .
therefore , tests for sps should be used for surveillance , especially in an outbreak in 
 fmd - free countries , because tests for sps may be able to detect fmdv infections earlier than 
 tests for nsps . in our previous study ,
the sensitivities of 
 three nsp - elisa kits were significantly low in infected animals without vaccination tested 
 in the 2010 epidemic in japan .
in contrast to our results , the sensitivities of several 
 nsp - elisa kits were reported to be high in infected animals without vaccination [ 2 , 3 , 5 , 16 ] .
both of the 
 results were completely inconsistent with each other . in our previous study , the antibody 
 titers of serum samples in the lpbe were the focus of inquiry regarding the inconsistent 
 results , because precise dates when the animals had been infected with the fmdv in the field 
 were unknown .
serum samples that showed relatively high antibody titers were speculated to 
 have been collected from animals a longer time after virus infection .
however , many serum 
 samples that showed relatively high antibody titers in the lpbe showed negative results in 
 the nsp - elisa kits . to explore the reasons for this inconsistency , we focused on the 
 relationships between the results of the rt - pcr assay , lpbe and nsp - elisa kit in this 
 study .
as mentioned in tables 1 and 2 , and the chapter of the results of infected pigs by the 
 intranasal and intraoral routes without vaccination , basically , the viral genes by the 
 rt - pcr assay , the antibodies by the lpbe and the antibodies by the priocheck kit were 
 sequentially detected in infected animals without vaccination .
detection of the viral genes 
 by the rt - pcr assay therefore meant that the samples were collected early after the fmdv 
 infection .
in contrast , relatively high antibody titers did not usually mean that the 
 samples were collected late after the animals were infected with fmdv , because high antibody 
 titers were observed even at early dpi when the viral genes were detected simultaneously by 
 the rt - pcr assay .
from these results , early collection of samples after the animals were 
 infected with an fmdv therefore may be the reason for the significantly low sensitivities of 
 the nsp - elisa kits in the serum samples collected from the animals in the field in the 2010 
 epidemic in japan .
antibody responses were influenced by viral doses for infection in the pigs ( table 3 ) .
the antibodies in the lpbe were detected 
 earlier in pigs 134 to 137 inoculated with 10 tcid50 of the isolate 
 than in pigs 131 to 133 inoculated with 10 tcid50 of the isolate . in 
 the antibody titers ,
the serum sample collected from pig 133 at 13 dpi showed the highest 
 antibody titer , as 2048 .
the pig and pig 131 were assumed to be infected by horizontal 
 transmission from pig 132 .
therefore , high viral 
 dose infection from pig 132 may stimulate the highest antibody titer in pig 133 . in previous studies [ 2 , 3 , 5 , 16 ] ,
sensitivities of several evaluated nsp - elisa kits were relatively 
 high in infected animals without vaccination .
on the other hand , sensitivities of the 
 nsp - elisa kits were considerably low in infected animals with vaccination in the studies .
as 
 a reason for these differential sensitivities , it was speculated that viral replication is 
 limited in infected animals with vaccination and that serological conversion to nsps occurs 
 more slowly and in a lower proportion than in infected animals without vaccination .
however , 
 the precise reason why there are cases in which antibodies to nsps are not detected in 
 infected animals with vaccination has not yet been confirmed .
a report proposed that studies 
 should be encouraged to define pathogenicity including antibody responses to nsps in 
 infected animals with vaccination . in this study , 

antibodies were detected fully from serum samples collected from infected cattle with 
 vaccination by the priocheck kit , although the first detection dates were later than those 
 in the lpbe ( table 
 4 ) .
the priocheck kit therefore may be applicable for serological 
 surveillance in cattle after emergency vaccination would be practiced in an fmd outbreak . 

however , it should be noted that tests for nsps are generally considered reliable at a herd 
 level [ 1 , 16 ] . 

in addition , antibody responses to nsps in infected cattle with vaccination need to be 
 further evaluated in different experimental conditions , such as viral infected doses and 
 routes . on the other hand , the study of the infected pigs with vaccination showed the different 
 results .
the priocheck kit did not detect antibodies in pig 1 during the experimental 
 period , and in pig 2 , it detected antibodies at only 7 dpi , although both the pigs were 
 confirmed to be infected with the fmdv by the results of the detection of the viral genes 
 from the clinical samples ( table 5 ) . 

well - vaccinated animals may become subclinically infected , even if they are exposed to a 
 sufficient virus challenge [ 8 , 15 ] .
similarly , in this study , pigs 1 to 4 did not show any clinical 
 signs , even though viral infection occurred ( table 
 5 ) .
the priocheck kit therefore may not be suitable to apply for serological 
 surveillance in pigs after emergency vaccination would be practiced in an fmd outbreak , 
 because infected pigs with vaccination may not be detected by either serological or clinical 
 surveillance .
if the priocheck kit would be applied as an assay for serological surveillance 
 in pigs after emergency vaccination would be practiced in an fmd outbreak , statistical 
 sufficient numbers of pigs should be examined by the kit .
in addition , a high sensitive 
 assay , such as an rt - pcr assay , should be applied simultaneously , because other pigs , which 
 are in an acute stage of infection , may exist in the same premise .
however , rapid sampling 
 from the large numbers of pigs would be hard to perform during an outbreak .
to reveal 
 availability for serological surveillance in pigs , the priocheck kit needs to be further 
 evaluated using infected pigs with vaccination before it is used for surveillance purposes . 
 in addition ,
evaluation of these two elisa kits for antibody detection in infected goats 
 with vaccination might be needed , although goats along with sheep are not major domestic 
 animals in japan . in conclusion ,
the following was confirmed in this study : ( i ) the lpbe can detect 
 antibodies in infected animals without vaccination earlier than the priocheck kit ; ( ii ) the 
 significantly low sensitivities of the nsp - elisa kits in the previous study may have been 
 due to the early collection dates of samples after animals were infected with fmdv ; and 
 ( iii ) the priocheck kit may not be able to detect antibodies in infected pigs with 
 vaccination
. these results will be valuable for authorities in choosing adequate control 
 measures for possible fmd outbreaks in the future .
 an elisa kit for detection of antibodies to a nonstructural protein of foot - and - mouth 
 disease ( fmdv ) was further evaluated using sequentially collected serum samples of 
 experimentally infected animals , because the sensitivity of the kit used in a previous 
 study was significantly low in field animals . 
 the kit fully detected antibodies in 
 infected animals without vaccination ; however , the first detections of antibodies by the 
 kit were later than those by the liquid - phase blocking elisa that is used for serological 
 surveillance in the aftermath of outbreaks in japan , for detection of antibodies to 
 structural proteins of fmdv . 
 additionally , although the kit effectively detected 
 antibodies in infected cattle with vaccination , there were several infected pigs with 
 vaccination for which the kit did not detect antibodies during the experimental period . 
 
 taken together , the kit may not be suitable for serological surveillance after an fmd 
 outbreak either with or without emergency vaccination in fmd - free countries . 
MATERIALS AND METHODS
RESULTS
DISCUSSION