toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds , causing toxoplasmosis .
t. gondii is an important foodborne parasite that is primarily transmitted from animals to humans through the consumption of infected meat . in some countries ,
pork is the most common meat consumed , and several ethnic groups consume raw pork .
toxoplasmosis is a source of significant economic loss for swine farmers because of gross lesions in infected animals , which result in the carcass being condemned at the time of slaughter , the expense associated with treatment , and weight loss associated with clinical toxoplasmosis .
the development of effective diagnostic reagents or vaccines is very important for worldwide public health and economic repercussions of t. gondii infection .
the life cycle of t. gondii is relatively complex , and its antigenic component can change in specificity or makeup during different development stages ; therefore , the newly synthesized multiepitope antigen is one of the most promising antigens for the development of effective diagnostic reagents or vaccines [ 4 - 9 ] .
however , the study of epitope - based vaccines and diagnostic reagents is highly dependent on the accurate identification of b - cell epitopes and t - cell epitopes .
therefore , the identification of protein epitopes will be very important for diagnostic purposes and for the development of peptide vaccines [ 10 - 12 ] . among dense granule antigens ( gras ) ,
gra6 was also demonstrated to be useful for designing novel and alternative diagnostic methods for toxoplasmosis or vaccines [ 13 - 17 ] .
the gra6 gene does not contain any introns and is a single copy in the genome of t. gondii .
gra6 localized in the dense granules and in the parasitophorous vacuole closely associated to the network .
a total of 51 t. gondii - positive sera samples previously collected from pigs ( n=32 ) experimentally infected with the gansu jingtai strain ( isolated from a pig with acute toxoplasmosis ) in our laboratory were evaluated in this study .
the experimental protocol was approved by the ethical committee of the lanzhou veterinary research institute , chinese academy of agricultural sciences , china .
twelve pig serum samples were collected at the time of presentation of clinical symptoms ( g1 ) , 18 follow - up samples were collected on days 14 to 35 after the onset of symptoms ( g2 ) , and 21 samples were collected on days 60 to 120 after the onset of symptoms ( g3 ) .
the presence of toxoplasma igm and igg antibodies was determined by t. gondii lysate antigen - elisa .
t. gondii dna was obtained from gansu jingtai strain tachyzoites using the universal genomic dna extraction kit ( takara biotechnology co. , ltd , dalian , china ) , and the gra6 sequence was amplified using the primers 5-gcgaattcatggcacacggtggcatct-3  and 5-atgcggccgcttaaaaatcaaactcattc-3  .
the pcr amplification was performed using the takara taqtm kit according to the manufacturer s instructions .
the sample was subjected to an initial denaturation ( 94c for 5 min ) , 35 cycles of denaturation ( 94c for 1 min ) , annealing ( 60c for 30 sec ) and elongation ( 72c for 1 min ) , and a final extension at 72c for 10 min .
the pcr - generated fragment was purified and cloned into the pmd-18 t vector ( takara biotechnology ) .
the recombinant plasmid was used to transform escherichia coli jm 109 competent cells , and the recombinant cells were selected on lb plates with ampicillin ( 100 mg / l ) , x - gal ( 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ; 70 mg / l ) , and iptg ( isopropyl -d - thiogalactopyranoside ; 80 m ) at 37c for 24 hr ( ampicillin , x - gal and iptg were from takara biotechnology ) .
positive colonies were inoculated into lb liquid medium containing ampicillin ( 100 mg / l ) and incubated at 37c for 16 hr .
the positive colonies identified by pcr were sequenced by takara biotechnology . to analyze the gra6 b cell epitopes ,
the deduced amino acid sequence of gra6 was analyzed using the protean subroutine in the dnastar software package .
this subroutine uses the garnier - robson   and chou - fasman   algorithms for predicting the alpha , beta , and turn regions , the garnier - robson algorithm for predicting the coil regions , the kyte - doolittle   algorithm for predicting hydrophilicity , the karplus - schultz   algorithm for predicting flexibility , the emini   algorithm for predicting surface probability , and the jameson - wolf   algorithm for predicting antigenicity .
based on this analysis , the peptides with good hydrophilicity , high accessibility , high flexibility , and strong antigenicity were selected as the antigen epitopes .
enzyme - linked immunoassays specific for each peptide were performed as described by cardona et al .   with minor modifications .
the microplates were coated with 100 l ( 10 g / ml ) of each peptide diluted in carbonate buffer , ph 9.6 ( na2co3 : 0.159 g/100 ml ; nahco3 : 0.293 g/100 ml ) .
the plates were incubated for 1 hr at 37c , then for 48 hr at 4c , and 1 hr at 37c .
non - specific ligand sites were blocked with 100 l 2% casein phosphate buffer for 1 hr at 37c . the plates were washed and incubated with 100 l serum diluted to 1:100 in 5% casein phosphate buffer for 1 hr at 40c .
after washing , 100 l rabbit anti - pig peroxidase - conjugated igg ( sigma ) diluted to 1:4,000 in 6% casein phosphate buffer was added for 20 min at 37c .
after the washes , the horseradish peroxidase activity was detected using tmb for 30 min at 37c and stopped with a 5% h2so4 solution .
a positive cut - off point was determined by estimating the mean average absorbance of 10 negative controls plus 2 sds . to determine the specificity of the anti - peptide antibody , elisa using irrelevant peptides from the bl21 of the orf virus previously synthesized by our laboratory ( sequence : vdvqskdkdadelre )
was also performed as described above . as controls , elisa using excretory / secretory antigen ( esa ) and recombinant gra6 from the rh strain of t. gondii previously expressed by our laboratory
briefly , non - specific ligand sites were blocked with 100 l 5% bsa phosphate buffer .
the rabbit anti - pig peroxidase - conjugated igg diluted 1:8,000 in pbs was used as the secondary antibody .
a total of 51 t. gondii - positive sera samples previously collected from pigs ( n=32 ) experimentally infected with the gansu jingtai strain ( isolated from a pig with acute toxoplasmosis ) in our laboratory were evaluated in this study .
the experimental protocol was approved by the ethical committee of the lanzhou veterinary research institute , chinese academy of agricultural sciences , china .
twelve pig serum samples were collected at the time of presentation of clinical symptoms ( g1 ) , 18 follow - up samples were collected on days 14 to 35 after the onset of symptoms ( g2 ) , and 21 samples were collected on days 60 to 120 after the onset of symptoms ( g3 ) .
the presence of toxoplasma igm and igg antibodies was determined by t. gondii lysate antigen - elisa .
t. gondii dna was obtained from gansu jingtai strain tachyzoites using the universal genomic dna extraction kit ( takara biotechnology co. , ltd , dalian , china ) , and the gra6 sequence was amplified using the primers 5-gcgaattcatggcacacggtggcatct-3  and 5-atgcggccgcttaaaaatcaaactcattc-3  .
the pcr amplification was performed using the takara taqtm kit according to the manufacturer s instructions .
the sample was subjected to an initial denaturation ( 94c for 5 min ) , 35 cycles of denaturation ( 94c for 1 min ) , annealing ( 60c for 30 sec ) and elongation ( 72c for 1 min ) , and a final extension at 72c for 10 min .
the pcr - generated fragment was purified and cloned into the pmd-18 t vector ( takara biotechnology ) .
the recombinant plasmid was used to transform escherichia coli jm 109 competent cells , and the recombinant cells were selected on lb plates with ampicillin ( 100 mg / l ) , x - gal ( 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ; 70 mg / l ) , and iptg ( isopropyl -d - thiogalactopyranoside ; 80 m ) at 37c for 24 hr ( ampicillin , x - gal and iptg were from takara biotechnology ) .
positive colonies were inoculated into lb liquid medium containing ampicillin ( 100 mg / l ) and incubated at 37c for 16 hr .
to analyze the gra6 b cell epitopes , the deduced amino acid sequence of gra6 was analyzed using the protean subroutine in the dnastar software package .
this subroutine uses the garnier - robson   and chou - fasman   algorithms for predicting the alpha , beta , and turn regions , the garnier - robson algorithm for predicting the coil regions , the kyte - doolittle   algorithm for predicting hydrophilicity , the karplus - schultz   algorithm for predicting flexibility , the emini   algorithm for predicting surface probability , and the jameson - wolf   algorithm for predicting antigenicity .
based on this analysis , the peptides with good hydrophilicity , high accessibility , high flexibility , and strong antigenicity were selected as the antigen epitopes .
enzyme - linked immunoassays specific for each peptide were performed as described by cardona et al .   with minor modifications .
the microplates were coated with 100 l ( 10 g / ml ) of each peptide diluted in carbonate buffer , ph 9.6 ( na2co3 : 0.159 g/100 ml ; nahco3 : 0.293 g/100 ml ) .
the plates were incubated for 1 hr at 37c , then for 48 hr at 4c , and 1 hr at 37c .
non - specific ligand sites were blocked with 100 l 2% casein phosphate buffer for 1 hr at 37c .
the plates were washed and incubated with 100 l serum diluted to 1:100 in 5% casein phosphate buffer for 1 hr at 40c .
after washing , 100 l rabbit anti - pig peroxidase - conjugated igg ( sigma ) diluted to 1:4,000 in 6% casein phosphate buffer was added for 20 min at 37c .
after the washes , the horseradish peroxidase activity was detected using tmb for 30 min at 37c and stopped with a 5% h2so4 solution .
a positive cut - off point was determined by estimating the mean average absorbance of 10 negative controls plus 2 sds . to determine the specificity of the anti - peptide antibody , elisa using irrelevant peptides from the bl21 of the orf virus previously synthesized by our laboratory ( sequence : vdvqskdkdadelre )
was also performed as described above . as controls , elisa using excretory / secretory antigen ( esa ) and recombinant gra6 from the rh strain of t. gondii previously expressed by our laboratory
briefly , non - specific ligand sites were blocked with 100 l 5% bsa phosphate buffer .
the rabbit anti - pig peroxidase - conjugated igg diluted 1:8,000 in pbs was used as the secondary antibody .
the secondary structure of gra6 was predicted by the garnier - robson and chou - fasman algorithms based on the sequence of the gra6 gene .
a flexibility plot , hydrophilicity plot , surface probability plot , and antigenic index for gra6 were obtained using the karplus - schulz , kyte - doolittle , emini , and jameson - wolf algorithms , respectively ( fig .
1 ) . based on the results obtained with these methods , potential b cell epitopes on gra6 were predicted , including 1 - 20 aa , 44 - 63 aa , 54 - 73 aa , 64 - 83 aa , 74 - 93 aa , 84 - 103 aa , 172 - 191 aa , 182 - 201 aa , 192 - 211 aa , and 202 - 221 aa .
all of the 10 predicted epitope peptides were evaluated by elisa using pig sera from various time points after infection .
the results of elisa for 3 peptides , p2 , p10 , and p11 , are shown in fig .
no significant differences were found between the mean absorbances of the 3 groups ( g1 , g2 , and g3 ) as determined by anova .
furthermore , no significant differences were found between the mean absorbances of the 3 peptides , p2 , p7 , and p9 .
the other 7 peptides were recognized by selection of sera from various time points after infection ( fig .
the number of positive samples / tested for each peptide was as follows : p1:8/51 , p3:25/51 , p4:18/51 , p5:9/51 , p6:27/51 , p8:20/51 , and p10:31/51 . to determine the specificity of the anti - peptide antibody , elisa using an irrelevant peptide
4a ) . to compare the serological reactivity of the peptides with esa and recombinant gra6 , elisa using esa and recombinant gra6
the adoption of immunoinformatics methods for the prediction of antigenic epitopes has become an indispensable tool for epitope localization .
in addition , such techniques are economical and effective and can substantially reduce experimental costs .
bioinformatics has been widely used in the analysis of protein epitopes [ 10 - 12 ] . in the present study ,
the secondary structure of gra6 was predicted by the garnier - robson and chou - fasman algorithms based on the sequence of the gra6 gene .
a flexibility plot , hydrophilicity plot , surface probability plot , and antigenic index for gra6 were obtained using the karplus - schulz , kyte - doolittle , emini , and jameson - wolf algorithms , respectively .
the existence of flexible regions , such as coil and turn region , provides powerful evidence for epitope identification . in the past ,
several experimental techniques were developed for mapping antibody interacting residues on an antigen , including the identification of interacting residues from the structure of antibody - antigen complexes .
many researchers have applied this technique to study epitopes [ 6,10 - 12,26,29 ] . using this technique and bioinformatics tools
, we found that many regions of gra6 , particularly the regions represented by peptides p2 , p7 , and p9 , are involved in the pig antibody response , and strong reactivity with the t. gondii - infected pig sera was observed .
the reactivity of these epitopes does not seem to be dependent upon the time of infection .
the identification of b cell epitopes is important for understanding antigenic structure and parasite - antibody interactions at the molecular level and may assist in the design of vaccines and diagnostic reagents .
conformational epitope selection relies on the determination of the tertiary structure of an antigen to identify residues that interact with antibodies .
the experimental techniques required to determine the tertiary structure of the antigen , such as crystallography , are expensive and time - consuming , and the mapping of conformational epitopes has been severely hampered .
the majority of methods and databases have focused on the identification of linear epitopes . in the present study
, linear epitopes were analyzed using synthetic peptide techniques and bioinformatics tools , and 3 of the 10 predicted epitope peptides were confirmed by synthetic peptide techniques .
the use of a molecular biology method in combination with a bioinformatics method is a useful method to screen for linear epitopes .
we have precisely located the epitopes of t. gondii gra6 using pig sera collected at different time points after infection .
the identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of epitope - based vaccines and diagnostic methods .
there are more linear and conformational b cell epitopes than previously predicted ; therefore , the number of identified epitopes should also increase with further studies .
 the study of antigenic epitopes from toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens , the reactions between antigens and antibodies , and many other aspects of immunology , but it also plays a significant role in the development of new diagnostic reagents and vaccines . in the present study , t. gondii gra6 epitopes were identified using bioinformatics tools and a synthetic peptide technique . 
 the potential b cell epitopes of gra6 predicted by bioinformatics tools concentrated upon 3 regions of gra6 , 1 - 20 aa , 44 - 103 aa , and 172 - 221 aa . 
 ten shorter peptides from the 3 regions were synthesized and assessed by elisa using pig sera from different time points after infection . 
 three of the 10 peptides ( amino acids 44 - 63 , 172 - 191 , and 192 - 211 ) tested were recognized by all sera and determined to be immunodominant b - cell epitopes of gra6 . 
 the results indicated that we precisely and accurately located the t. gondii gra6 epitopes using pig sera collected at different time points after infection . 
 the identified epitopes may be very useful for further studies of epitope - based vaccines and diagnostic reagents . 