samples : serum samples were taken from 337 pet rabbits presented to 34 
 veterinary facilities in different 20 prefectures in japan . healthy rabbits were brought to the veterinary clinics for either general 
 health checks or other disease conditions . among the rabbits , the rest showed variable clinical 
 conditions , such as embraced tumors or disorders of the digestive , respiratory or urinary 
 systems . the examined rabbits 
 ranged in age from 3 months to 10 years old , and the average age was 3.4 years . the breeds 
 of the tested rabbits and their numbers were as following : 16 holland lop , 28 netherland 
 dwarf , 3 lion , 9 angora , 3 rex , 2 dutch , 1 american fuzzy lop , 1 jersey wooly , 272 others 
 and 2 unknown rabbits . all rabbit serum samples referred to our laboratory had been tested 
 to assess the seroprevalences of t. gondii and n. caninum 
 antibodies . parasites : t. gondii ( rh and me49 
 strains ) and n. caninum ( nc1 strain ) tachyzoites were maintained in human 
 foreskin fibroblast ( hff ) cells cultured in dulbecco s modified eagle s medium ( dmem , gibco , 
 grand island , ne , u.s.a . ) supplemented with 7.5% heat - inactivated fetal bovine serum ( fbs ) . 
 for purification and lysis of tachyzoites , the infected cells were washed with cold 
 phosphate - buffered saline ( pbs ) . cell pellets were resuspended in medium and passed through 
 a 27-gauge needle and then through a 5.0 m - pore filter ( millipore , 
 bedford , ma , u.s.a . ) . after centrifugation at 2,000  g for 5 min at 4c , 
 the pellet was resuspended with ripa buffer ( 50 mm tris - hcl , ph 7.5 , 150 mm nacl , 1 mm edta , 
 0.25 mm sodium deoxycholate , 0.1% triton x-100 and 1% nonidet p-40 ) . the tachyzoite lysates 
 were recovered after centrifugation at 2,000  g for 5 min . recombinant protein production : recombinant proteins of t. 
 gondii surface antigen 2 ( tgsag2 ) and n. 
 caninum surface antigen 1 ( ncsag1 ) were generated according to 
 previous reports [ 20 , 21 ] . generation of polyclonal anti - tgsag2 antisera : polyclonal 
 anti - tgsag2 antisera were produced in balb / c mice . immunization of mice 
 was performed as described previously . in brief , 
 mice were immunized subcutaneously at three different inoculation sites with 10 
 g of recombinant tgsag2 protein emulsified with an 
 equal volume of freund s complete adjuvant . two weeks later , mice were immunized with the 
 same dose of antigen emulsified with freund s incomplete adjuvant . another dose of the 
 antigen with the incomplete adjuvant was given on day 28 . seven days later , mice were 
 sacrificed under isoflurane anesthesia , and blood was collected for serum extraction . the experiments were 
 conducted according to the guidelines for the care and use of laboratory animals issued by 
 obihiro university of agriculture and veterinary medicine . elisa : one hundred microliters of antigen ( either tachyzoites lysate or 
 recombinant protein ) in a coating buffer ( 50 mm carbonate , ph 9.6 ) at concentration of 2 
 g / ml were coated onto elisa plates ( nunc , roskilde , 
 denmark ) and incubated overnight at 4c . plates were washed 5 times with pbs containing 
 0.05% tween 20 ( pbs - t ) and blocked with pbs containing 1% skimmed milk ( pbs - sm ) for 1 hr at 
 room temperature ( rt ) . after washing with pbs - t , 100 l of rabbit serum 
 diluted at 1:500 with pbs - sm were added to duplicate wells and incubated for 1 hr at rt . 
 after washing 5 times with pbs - t , the plates were incubated with horseradish peroxidase 
 ( hrp)-conjugated anti - rabbit igg ( ge healthcare uk limited , buckinghamshire , u.k . ) or igm 
 ( southernbiotech , birmingham , al , u.s.a . ) diluted at 1:2,000 with pbs - sm at rt for 1 hr . plates were further washed 5 times before the substrate solution [ 0.1 m citrate buffer , ph 
 4 , 0.003% h2o2 and 0.3 mg / ml 2 , 2-azino - bis 
 ( 3-ethylbenzothiazoline-6-sulfonic acid ) ; sigma - aldrich ] was added to each well in 100 
 l aliquots . the absorbance at 415 nm after 30 min of incubation at rt 
 was measured using an elisa reader ( corona microplate reader mtp-120 ; corona , tokyo , japan ) . the cutoff point was determined as the average value of seronegative rabbit samples ( n=24 ) 
 plus 3 sd . latex agglutination test : the lat was performed according to the kit 
 manufacturer s instructions ( toxocheck - mt , eiken chemical , tokyo , japan ) . samples were 
 considered positive when agglutination was observed at a dilution of 1:32 or greater . western blot analysis : lysates of t. gondii ( rh strain ) 
 and n. caninum were dissolved in sodium dodecyl sulfate polyacrylamide gel 
 electrophoresis ( sds - page ) sample buffer ( 62.5 mm tris - hcl , ph 6.8 , 2% sds , 140 mm 
 2-mercaptoethanol , 10% glycerol and 0.02% bromophenol blue ) heated at 95c for 5 min and 
 separated on a 12% polyacrylamide gel . all separated proteins were electrically transferred 
 onto polyvinylidene fluoride ( pvdf ) membranes ( immobilon - p transfer membrane , millipore , 
 billerica , ma , u.s.a . ) using a western blot apparatus ( horizeblot type ae-6677 , atto 
 bioscience & biotechnology , tokyo , japan ) . after blocking for 1 hr in pbs - sm , membranes 
 were probed with the rabbit serum diluted at 1:500 with pbs - sm for 1 hr . the membranes were 
 washed 3 times for 10 min with pbs - t and then probed with hrp - conjugated anti - rabbit igg 
 diluted at 1:2,000 in pbs - sm for 1 hr at rt . after washing with pbs - t , the membranes were 
 immersed in the detection solution ( 0.1 m tris , ph 8.5 , 1.25 mm luminol acid , 0.2 mm 
 coumaric acid and 0.075% h2o2 ) and exposed to x ray film . prestained 
 molecular mass standards ( genedirex , biospeed , las vegas , nv , u.s.a . ) were used . immunoprecipitation : one hundred micrograms of t. gondii 
 lysate were mixed with 2 l of either seronegative or seropositive rabbit 
 serum and kept on ice for 1 hr . twenty microliters of protein g sepharose ( ge healthcare , 
 uppsala , sweden ) were added to each sample and rotated at 4c for 1 hr . beads were washed 5 
 times with pbs , and then protein samples were separated on 12% polyacrylamide gel and 
 electrically transferred onto pvdf membranes . , long beach , ca , 
 u.s.a . ) or mouse polyclonal anti - sag2 antibodies were used to probe the membranes at a 
 dilution of 1:500 in pbs - sm . after washing 3 times with pbs - t , membranes were probed with 
 hrp - conjugated anti - mouse igg diluted at 1:2,000 in pbs - sm incubated for 1 hr at rt . indirect immunofluorescence assay ( ifa ) : the indirect immunofluorescence 
 was performed as described previously   with some 
 modifications . glass coverslips with confluent monolayers of hff cells were infected with 
 t. gondii ( me49 strain ) and incubated in a co2 incubator for 
 24 hr at 37c . after washing 3 times with pbs , cells were fixed and permeabilized with 4% 
 formaldehyde and 0.2% triton x-100 in pbs ( ph 7.2 ) for 15 min . cells were then washed 3 
 times with pbs and blocked for 30 min in 3% bsa / pbs . coverslips were incubated with rabbit 
 serum samples diluted at 1:100 , 1:500 and 1:1,000 in 3% bsa / pbs . after washing 3 times with pbs , coverslips were incubated with alexa fluor 594-conjugated 
 goat anti - rabbit igg and alexa fluor-488 conjugated goat anti - mouse igg ( invitrogen ) diluted 
 at 1:1,000 in 3% bsa / pbs . mowiol ( 4.8 g mowiol , 12 g glycerol in 50 mm tris hcl , ph 8.5 ) was used as a mounting 
 medium . samples were examined on a leica tcs nt confocal laser scanning microscope ( leica 
 microsystems , wetzlar , germany ) using the appropriate settings . green and red fluorescence 
 and differential interference contrast ( dic ) images were recorded using the leica powerscan 
 software . statistical analysis : the results of the different serological tests were 
 estimated by the percentage of agreement , the sensitivity and specificity and the kappa 
 values using ( http://vassarstats.net/ ) . detection of specific antibodies raised by natural infections of t. gondii 
 or n. caninum : to assess the presence of toxoplasmosis or neosporosis in 
 pet rabbits , igg and igm elisa tests were carried out to screen all rabbit sera ( 337 ) using 
 tachyzoites lysate of t. gondii or n. caninum and 
 tgsag2 or ncsag1 recombinant protein as antigens . seropositive samples were further examined by lat , western blotting and ifa . for t. gondii , igg - elisa identified 9 positive sera according to the calculated cutoff 
 value ( 0.6 ) , 6 samples scored an od value below 1 , 1 sample scored an od value between 1 and 
 2 , and the remaining 2 sera had od values of more than 2 . only one sample was found to be 
 positive in the igm elisa ( table 1table 1.results of the serological tests performed on sera from pet rabbitstested parasiteno . of rabbitselisa iggelisa igmlatwestern blotifaabs ( od)abs ( od)0.6112 20.65112  2 t . gondii337612001333n . all rabbit sera that showed positive reactivity with lysate antigen were 
 also reactive with the recombinant protein . sample that scored od values below 0.6 ( 328 ) 
 were considered negative by elisa test . d ) ifa : indirect immunofluorescence antibody assay . to include a wide range of samples , the latex agglutination test was applied to all samples 
 that showed an od value above 0.3 ( 35/337 ) using the recommended dilutions . agglutination 
 was observed only in 3 samples ( od value above 1 ) at dilutions of 1:2,048 and 1:128 ( table 1 ) , while the remaining 32 samples did not 
 show any agglutination and were therefore considered negative . in addition , all samples that 
 showed od values above the elisa cutoff point ( 0.6 ) were analyzed by western blotting and 
 ifa . both tests resulted in 3 positive sera that had shown positivity also in the lat ( fig . a ) lanes 1 , 2 and 3 represent seropositive rabbit samples for 
 t. gondii . the analysis 
 was performed by using an equal volume of purified t. gondii 
 tachyzoite lysate of the me49 strain as the antigen . b ) specific bands of sag1 can be 
 observed at 30 kda ( left panel ) , and those of sag2 can be observed at 22 kda ( right 
 panel ) . lane 2 shows mouse anti - sag1 monoclonal ( left panel ) and mouse 
 anti - sag2 polyclonal ( right panel ) antibodies , respectively . 2.immunoprecipitates of either seronegative ( lane 2 ) or seropositive ( lane 3 ) samples 
 reacted with either the mouse anti - sag1 monoclonal antibody ( left panel ) or mouse 
 anti - sag2 polyclonal antibody ( right panel ) . the black arrow represents the specific band of sag1 ( left ) and sag2 ( right ) 
 precipitated only by the positive sera . arrowheads represent igg heavy ( upper ) and 
 light ( lower ) chains , respectively . rows 1 , 2 and 3 represent t. gondii - seropositive 
 samples ( left panel , red ) . the mouse 
 anti - t . gondii imc1 antibody ( green ) was used for 
 counterstaining . we found that the only serum samples that scored elisa od values higher 
 than 1.0 were detectable by lat , western blot and ifa ( 3/337 ) . a ) lanes 1 , 2 and 3 represent seropositive rabbit samples for 
 t. gondii . the analysis 
 was performed by using an equal volume of purified t. gondii 
 tachyzoite lysate of the me49 strain as the antigen . b ) specific bands of sag1 can be 
 observed at 30 kda ( left panel ) , and those of sag2 can be observed at 22 kda ( right 
 panel ) . lane 1 shows seropositive rabbit sample naturally infected with t. 
 gondii . lane 2 shows mouse anti - sag1 monoclonal ( left panel ) and mouse 
 anti - sag2 polyclonal ( right panel ) antibodies , respectively . immunoprecipitates of either seronegative ( lane 2 ) or seropositive ( lane 3 ) samples 
 reacted with either the mouse anti - sag1 monoclonal antibody ( left panel ) or mouse 
 anti - sag2 polyclonal antibody ( right panel ) . the black arrow represents the specific band of sag1 ( left ) and sag2 ( right ) 
 precipitated only by the positive sera . arrowheads represent igg heavy ( upper ) and 
 light ( lower ) chains , respectively . rows 1 , 2 and 3 represent t. gondii - seropositive 
 samples ( left panel , red ) . gondii imc1 antibody ( green ) was used for 
 counterstaining . on the other hand , when rabbit samples tested for the presence of n. 
 caninum antibodies , although igg - elisa showed 10 cases over the cutoff value 
 ( 0.6 ) , 9 of them had od values below 1 , and one case had an od value between 1 and 2 ( table 1 ) . no specific band or reactive staining was 
 observed for n. caninum in both western blot and the ifa . detection of specific antigens in sera of naturally infected rabbits with t. 
 gondii : in western blotting , positive sera generated multiple distinct bands 
 between 14 and 130 kda ( fig . certain protein 
 bands were noticeably common in all positive sera at 30 and 22 kda , the same molecular 
 weights as the sag1   and sag2   antigens . to confirm this , western blot analyses were 
 performed to compare the sizes of identified bands in the sera of infected rabbits with the 
 mouse anti - sag1 monoclonal and anti - sag2 polyclonal antibodies . further 
 confirmation was performed by immunoprecipitation using seropositive and seronegative 
 t. gondii rabbit serum samples that were tested previously by elisa and 
 lat . the staining pattern in the ifa of positive 
 sera showed a strong signal on the surface of the parasites ( fig . 3 ) . the results of elisa , ifa and western blot were statistically calculated to obtain the 
 specificity and sensitivity and the kappa value to assess their ability to detect t. 
 gondii antibodies in rabbit sera in comparison with the lat as a reference test 
 ( table 2table 2.specificity and sensitivity of the elisa , ifa and western blot in detecting 
 t. gondii - specific antibodies in rabbit sera compared with the 
 results in the reference lat testparametervalue of performed serological tests compared with 
 the latelisaifawestern blotsensitivity ( % ) 100 ( 9/3)100 ( 3/3)100 ( 3/3)specificity ( % ) 98 ( 328/334)100 ( 334/334)100 ( 334/334)concordance ( % ) 98.2100100kappa value0.4911 ) . rabbits are especially popular as pets in japan ; dogs and cats may be the most frequent 
 household animals , but the popularity of rabbits is increasing . therefore , their health 
 status is also important to consider . moreover , questions often arise concerning the hazards 
 in the surrounding environments for both pet rabbits and the people around them . t. 
 gondii and n. caninum are protozoan parasites known to infect 
 humans and animals [ 12 , 37 ] . human beings can be infected with t. gondii and 
 possibly with n. caninum [ 16 , 28 , 38 ] by 
 ingestion , the major route , of either oocysts in contaminated food and water or meat 
 containing the parasite cysts . the ability to distinguish whether the original infection was 
 by tissue cyst or oocysts and discover whether the outcomes are different is a great concern 
 nowadays . because cats and dogs are so abundant in the environment and indeed are 
 increasing , contamination of the human environment with oocysts will continue to increase , 
 and exposure will be unavoidable . furthermore , recent studies reported that the majority of 
 congenital infections and postnatal acute infections in the united states originated from 
 oocysts , and this will increase the concern about 
 the severity and outcomes of infections primarily induced by oocysts . because the oocyst is 
 the only source of infection in rabbits , pet rabbits are considered good indicators for 
 assessment of the oocyst contamination in the human environment . therefore , in view of the 
 worldwide importance of these parasites and their infective stages , the seroprevalences of 
 both t. gondii and n. caninum in pet rabbits were 
 estimated in this study . serodiagnostic tools are important methods for detecting parasitic infections ; one of these 
 tools is the elisa using tachyzoite - derived antigens or generated recombinant proteins 
 [ 7 , 19 ] . in 
 this study , igg and igm elisas were used to analyze all rabbit sera by using the whole 
 tachyzoites - derived antigen of either t. gondii or n. 
 caninum . the use of such antigens may produce false positive results due to 
 cross - reaction with other closely related parasites   or unknown reasons . to validate our results , elisas based on the recombinant 
 proteins tgsag2 and ncsag1 similar 
 data were obtained , indicating that in this case , the antigenicity of the recombinant 
 proteins did not show any difference compared with the lysates . therefore , it was necessary 
 to perform other tests , such as the lat , western blotting and ifa . these three tests 
 identified only three positive sera that previously showed od values > 1 in the igg 
 elisa . also , we identified sag1 and sag2 as major antigens detected by the positive rabbit sera . these findings were confirmed by western blotting and immunoprecipitation , as shown in the 
 results . although the tgsag2 recombinant proteins have already been 
 established as specific antigens for diagnosis of t. gondii via elisa 
 , our results confirmed that sag1 and sag2 can 
 be used as useful target antigens for specific testing of rabbit sera to detect t. 
 gondii infection by other serological methods . concerning n. 
 caninum antibodies in rabbit sera , although elisa detected 10/337 positive sera , 
 western blot and the ifa did not reveal any positivity . therefore , it could be plausibly accepted that toxoplasmosis was not common and that 
 neosporosis was not prevalent among pet rabbits in japan . additionally , in this study , we 
 compared the positivity of the results between the elisa and the other serological methods . the rabbit sera tested by elisa showed 2.67% positivity for t. gondii and 
 2.96% for n. caninum . however , the seropositivity was only 0.89% for 
 t. gondii and 0% for n. caninum using western blotting 
 or the ifa . therefore , it is reasonable to accept that using more than one method in 
 serodiagnosis is recommended for accurate investigation . since we used different tests here for serological detection , it was important to emphasize 
 the capability of each test . therefore , the results of elisa , ifa and western blot were 
 statistically compared with those of the lat as a reference test for detection of t. 
 gondii antibodies ( table 2 ) . among 
 the tests used , the elisa showed a higher positive rate of detection of t. 
 gondii - specific igg antibodies than the ifa and western blot ( table 1 ) . although elisa achieved very high sensitivity in 
 detection ( 100% ) , the six negative samples that were detectable by elisa and not by lat 
 resulted in 98% specificity for elisa . the kappa value of 0.49 could be interpreted as a 
 moderate level of agreement . while , the sensitivity and specificity of the ifa and western 
 blot were both 100% when the lat was used as a reference test associated with high agreement 
 indicated by maximum kappa value ( 1 ) as summarized in table 2 . therefore , we could conclude from this analysis that the lat , ifa and 
 western blot had similar capabilities for detection of t. gondii antibodies 
 in rabbit sera . however , the availability of these tests for rapid screening is not similar , 
 because the ifa and western blotting are more sophisticated techniques that might not be 
 applicable to large number of samples or as a field test . moreover , both tests were unable 
 to detect the antibody titer , while the lat is convenient , more applicable and provides the 
 advantage of both qualitative and quantitative assessment of the infection status . concerning n. caninum , the results of elisa and western blot showed 97% and 
 100% specificity , respectively , when the ifa was used as a standard test , while the 
 sensitivity could not be calculated ( data not shown ) . in reference to the seroprevalence of t. gondii in rabbits in different 
 countries , tested domestic rabbits reared on farms and in backyards and pet shops in 
 northern mexico showed that antibodies to t. gondii were found in 16.3% of 
 429 rabbits . in korea , a similar type of rabbits 
 was surveyed from breeding farms , and 10.6% of them showed t. 
 gondii - specific igg antibodies . in 
 egypt , high seroprevalences of t. gondii were also detected , 37.5% and 
 11.34% , in locally bred and commercial rabbits . in 
 the studies described above , the high seroprevalences of t. gondii 
 infection could be correlated to factors , such as rearing outdoors or in a pet shop , feeding 
 of possibly contaminated vegetables and cohabitation with cats or dogs . additionally , considerably high prevalences for example , in china , the seroprevalence of t. 
 gondii antibodies was 23.4% in free domestic rabbits from a rural area . also , a study in spain   reported 14.2% t. gondii positivity among 456 wild 
 rabbits captured or hunted from forests . on the other hand , a study on wild cottontail 
 rabbits in northern italy mentioned low prevalences of both t. gondii and 
 n. caninum infections , 2.08% and 2.78% , respectively . in the present study , the seroprevalence of t. gondii was 0.89% in pet 
 rabbits of japan . referring to the previously mentioned studies performed on domestic or 
 wild rabbits , results can not be directly compared , although the susceptibilities were quite 
 similar because the rabbits were from the same species . it is important to consider the 
 surrounding environment and rearing conditions of the examined rabbits in this study . in 
 general , wild rabbits or even domestic rabbits that are reared or have access to the 
 outdoors might be more susceptible to such parasitic infections than pet rabbits confined 
 indoors , bearing in mind the risk of direct contact with the contaminated environment . in 
 japan , rabbits are considered pets and are mostly kept indoors or in cages as in a zoo . 
 although they are sometimes given time to range freely outside , this is different to some 
 extent from the case of domestic farm rabbits reared for meat purposes and to a great extent 
 from wild rabbits . given the unavailability of data concerning recovery of oocysts of the 
 aforementioned parasites from the human environment , little is known about their potential 
 hazards . in conclusion , the present study indicates that there is probably a low level of oocyst 
 contamination in the human environment according to estimation of the prevalences of 
 toxoplasmosis and neosporosis in pet rabbits . we concluded that t. gondii 
 was not common among healthy pet rabbits and those suffering from neurological disorders . furthermore , as far as could be checked , there were no apparent n. caninum 
 infections in pet rabbits . assessment of contamination of the surrounding environment with 
 oocysts of the parasites and its implications for humans and other animals requires further 
 attention and investigation .