BACKGROUND
toxoplasma gondii is an important zoonotic pathogen which can infect all warm-blooded animals and humans.
it is estimated that approximately one-third of human population in the world has been infected with t. gondii .
toxoplasmosis causes several clinical syndromes such as chorioretinitis, encephalitis and systemic infections, particularly seriously in pregnant hosts and immuno-compromised individuals such as those with hiv/aids .
also, toxoplasmosis can lead to considerable economic losses in livestock industry .
t. gondii isolates from different geographical locations and hosts have been grouped into  <dig> haplogroups that collectively define six major clades by pcr-restriction fragment length polymorphism   <cit> .
despite that genotyping t. gondii strains using polymorphisms has been focused on  <dig> markers including sag <dig>  sag <dig>  btub, gra <dig>  c29- <dig>  l <dig>  pk <dig>  5′-sag <dig>  3′-sag <dig>  alt.
sag <dig>  c22- <dig> and apico, additional gene  may contribute to some distinct t. gondii genotypes  <cit> .
the full knowledge of genetic diversity in t. gondii is a key to better understand pathogenicity and epidemiological patterns, and thus to explore a new way for vaccination, treatment and diagnosis of toxoplasmosis.
microneme proteins  of t. gondii play key roles in t. gondii survival and the invasion process, and thus affecting host cell signaling, as well as participating in the binding to host cell receptors .
among those mics, the microneme protein  <dig> of t. gondii  is responsible for binding to aldolase, is associated with rhomboid cleavage, and thus involved in the trafficking signals during invasion  <cit> .
although the major functions of tgmic <dig> were uncovered, little is known of the sequence variation in mic <dig> gene among t. gondii isolates.
therefore, the objectives of the present study were to examine sequence variation in mic <dig> gene among 12 t. gondii strains from different hosts and geographical locations, as well as to assess whether mic <dig> gene can provide a potential marker for population genetic studies of t. gondii by phylogenetic analysis and pcr-rflp.
methods
genomic dna samples of t. gondii
a total of  <dig> genomic dna samples of t. gondii were used in this study .
these t. gondii dna samples had been genotyped in our previous studies .
the dna samples of the reference strains gt <dig>  mas, tgcatbr <dig>  ptg, ctg and tgtoucan were kindly provided by associate professor chunlei su at the department of microbiology, the university of tennessee, knoxville, usa.table  <dig> details of toxoplasma gondii isolates used in this research
abased on the results of zhou et al.
<cit>  and su et al.
<cit>
pcr amplification
the entire genomic sequence of the mic <dig> gene was amplified by pcr using a pair of specific primers  designed based on the mic <dig> gene sequence of t. gondii gt <dig> strain .
the amplification reaction was carried out in a volume of 25 μl containing 10 mm tris–hcl , 50 mm kcl, 3 mm mgcl <dig>  250 μm each of dntp,  <dig>  μm of each primer, 100–200 ng of template dna, and  <dig>  u la taq polymerase .
amplification of dna samples from individual isolates was carried out in a thermocycler  under the following conditions: the initial denaturation at 95 °c for 5 min, followed by 34 cycles consisting of 95 °c for 1 min , 64 °c for 45 s , 72 °c for 4 min 30 s , and a final extension step was at 72 °c for 10 min.
each  amplification was carried out by electrophoresis on a 1 %  agarose gel with ml <dig> dna ladder marker , stained with goldenview™ and photographed using a gel documentation system   <cit> .
sequencing of the mic <dig> amplicons
to ensure the accuracy of mic <dig> sequences from different t. gondii strains, the pcr products of mic <dig> gene were purified according to manufacturer’s recommendations , and then ligated with the pgem-teasy .
thereafter, positive plasmids were transformed into the jm <dig> competent cells .
following pcr screening, the positive colonies were sequenced by genscript  co., ltd.
sequence analysis and phylogenetic reconstruction
the obtained mic <dig> gene sequences from different t. gondii strains were aligned using multiple sequence alignment program, clustal x  <dig>   <cit> , and sequence variation was determined among the examined t. gondii strains.
the phylogenetic reconstructions based on the mic <dig> sequences from different t. gondii strains were performed by bayesian inference  and maximum parsimony .
bi analyses were conducted with four independent markov chains run for  <dig> , <dig> metropolis coupled mcmc generations, sampling a tree every  <dig>  generations in mrbayes  <dig> .
<dig>  <cit> .
the first  <dig> trees were omitted as burn-in and the remaining trees were used to calculate bayesian posterior probabilities .
mp analysis was performed using paup*  <dig> b <dig>  <cit> .
bootstrap support for mp tree was calculated from  <dig> bootstrap replicates with  <dig> random additions per replicate  <cit> .
pcr-rflp
pcr-rflp was used to further assess whether mic <dig> gene sequence is a potential marker for genotyping t. gondii isolates.
the amplified mic <dig> fragments were digested with restriction enzyme pst i for 8 h at 37 °c in the appropriate buffer, and then inactivated at 80 °c for 20 min according to the manufacturer’s instructions .
the restriction fragments were analyzed by electrophoresis on 1 % agarose gel stained with goldenview™ and photographed using a gel documentation system .
RESULTS
sequence analysis
all the positive transformants of the mic <dig> gene were approximately 4000 bp in length on 1 %  agarose gel .
the mic <dig> gene sequences were 4391 bp in length for strains tgcatbr <dig> and mas; and they were 4394 bp in length for strains rh, tgplh, gt <dig>  pru, qho, ptg, pys, gjs, ctg and tgtoucan.
their a+t content varied from  <dig>  to  <dig>  %.
alignment of the obtained mic <dig> sequences from 12 t. gondii strains revealed nucleotide polymorphisms at  <dig> sequence variation positions , including  <dig> variations in  <dig> introns,  <dig> variations in  <dig> exons .
in addition, a total of  <dig> transversions ,  <dig> transitions ,  <dig> deletions and  <dig> inserts were identified in mic <dig> gene .
sequences analysis of polymorphisms in the three references genotypes  revealed the existence of polymorphic restriction sites.
using pcr-rflp method, digestion of the amplification products with pst i allowed the differentiation between genotype i, ii, and iii .fig.
<dig> phylogram of  <dig> toxoplasma gondii strains determined by analysis of the entire sequences of the mic <dig> gene.
the tree was built by bayesian inference  and maximum parsimony  analysis.
the numbers along branches show bootstrap values resulting from different analyses: bi/mp
phylogenetic analysis of t. gondii strains based on mic <dig> sequences
by phylogenetic reconstruction based on mic <dig> sequence data of all  <dig> strains, we have obtained the phylogenetic tree .
phylogenetic analysis revealed three major clusters, which are corresponding to classical genotypes  respectively, and type iii are clustered more closely with type i than with other strains.
atypical strains tgtoucan were phylogenetically linked to type iii, and thus the both strains gys and pys were phylogenetically linked to type ii.fig.
<dig> pcr-rflp analysis of mic <dig> genomic sequences of toxoplasma gondii isolates in 1 % agarose gel using restriction endonucleases pst i. lane m represents dna size marker  <dig>  lanes 1– <dig> represent t. gondii type i , type ii , type iii  strains respectively.
refer to table  <dig> for isolate information
discussion
in the present study, we determined the entire genome region of the mic <dig> locus for 12 t. gondii strains among different host and geographical origins, and determined genetic diversity between toxoplasma strains based on mic <dig> gene sequences.
it has been found that there were a total of  <dig> variable positions distributed in the genome sequence of the mic <dig> locus among  <dig> isolates, including  <dig> variations in  <dig> exons and  <dig> variations in  <dig> introns.
these results suggested higher sequence variations in mic <dig> gene than that in mic <dig>  mic <dig> and eif4a genes .
the analyses of mic <dig> gene in both nucleotides and amino acids among 12 t. gondii strains showed high ratio of non-synonymous to synonymous polymorphisms, emphasized that mic <dig> was undergoing positive selection together with some other loci, such as gra <dig>  gra <dig> and rop <dig>  <cit> .
with the purpose of analyzing the evolutionary relationship among the  <dig> examined t. gondii isolates, the phylogenetic tree was established  based on the mic <dig> sequence using bi and mp methods.
phylogenetic analysis showed that t. gondii strains corresponding to classical genotypes  were grouped into their respective clusters separately, which were consistent with the results from other genetic markers including gra <dig> and gra <dig> genes  <cit> .
moreover, the three major clonal lineages  can be differentiated by digestion of mic <dig> pcr products using endonuclease pst i , which further suggested that the mic <dig> locus may be a potential new genetic marker for pcr-rflp genotyping of t. gondii strains.
however, some atypical strains including tgcatbr <dig>  tgtoucan, pys, gjs and mas can not be distinguished from the three major lineage types by pcr-rflp , suggesting that pcr-rflp genotyping with a single locus is not enough for the identification of non-clonal genotypes.
CONCLUSIONS
the present study examined sequence variation in mic <dig> and demonstrated the existence of sequence variability in mic <dig> gene among the examined t. gondii strains from different hosts and geographical localities.
phylogenetic analysis and pcr-rflp genotyping of the examined t. gondii strains suggest that mic <dig> gene may provide a potential marker for population genetic studies of t. gondii isolates, and further research sampling more isolates from different hosts and geographical separations need to be done to verify this possibility.
abbreviations
aids, acquired immune deficiency syndrome; bi, bayesian inference; hiv, human immunodeficiency virus; mp, maximum parsimony; pcr, polymerase chain reaction; pcr-rflp, pcr-restriction fragment length polymorphism; tgmic <dig>  toxoplasma gondii microneme protein 16
additional file
additional file 1: figure s <dig>  alignment of mic <dig> gene sequences of  <dig> toxoplasma gondii strains.
lower case letter “a” indicates the base changes in exons; lower case letter “b” indicates the base changes in introns.

 toxoplasma gondii is an important zoonotic pathogen which can infect all warm-blooded animals and humans. it is estimated that approximately one-third of human population in the world has been infected with t. gondii. toxoplasmosis causes several clinical syndromes such as chorioretinitis, encephalitis and systemic infections, particularly seriously in pregnant hosts and immuno-compromised individuals such as those with hiv/aids. also, toxoplasmosis can lead to considerable economic losses in livestock industry. t. gondii isolates from different geographical locations and hosts have been grouped into haplogroups that collectively define six major clades by pcr-restriction fragment length polymorphism. despite that genotyping t. gondii strains using polymorphisms has been focused on markers including sag sag btub, gra c29- l pk 5′-sag 3′-sag alt. sag c22- and apico, additional gene may contribute to some distinct t. gondii genotypes. the full knowledge of genetic diversity in t. gondii is a key to better understand pathogenicity and epidemiological patterns, and thus to explore a new way for vaccination, treatment and diagnosis of toxoplasmosis. microneme proteins of t. gondii play key roles in t. gondii survival and the invasion process, and thus affecting host cell signaling, as well as participating in the binding to host cell receptors. among those mics, the microneme protein of t. gondii is responsible for binding to aldolase, is associated with rhomboid cleavage, and thus involved in the trafficking signals during invasion. although the major functions of tgmic were uncovered, little is known of the sequence variation in mic gene among t. gondii isolates. therefore, the objectives of the present study were to examine sequence variation in mic gene among 12 t. gondii strains from different hosts and geographical locations, as well as to assess whether mic gene can provide a potential marker for population genetic studies of t. gondii by phylogenetic analysis and pcr-rflp. methods genomic dna samples of t. gondii a total of genomic dna samples of t. gondii were used in this study. these t. gondii dna samples had been genotyped in our previous studies. the dna samples of the reference strains gt mas, tgcatbr ptg, ctg and tgtoucan were kindly provided by associate professor chunlei su at the department of microbiology, the university of tennessee, knoxville, usa.table details of toxoplasma gondii isolates used in this research abased on the results of zhou et al. and su et al. pcr amplification the entire genomic sequence of the mic gene was amplified by pcr using a pair of specific primers designed based on the mic gene sequence of t. gondii gt strain. the amplification reaction was carried out in a volume of 25 μl containing 10 mm tris–hcl, 50 mm kcl, 3 mm mgcl 250 μm each of dntp,  μm of each primer, 100–200 ng of template dna, and u la taq polymerase. amplification of dna samples from individual isolates was carried out in a thermocycler under the following conditions: the initial denaturation at 95 °c for 5 min, followed by 34 cycles consisting of 95 °c for 1 min, 64 °c for 45 s, 72 °c for 4 min 30 s, and a final extension step was at 72 °c for 10 min. each amplification was carried out by electrophoresis on a 1 % agarose gel with ml dna ladder marker, stained with goldenview™ and photographed using a gel documentation system. sequencing of the mic amplicons to ensure the accuracy of mic sequences from different t. gondii strains, the pcr products of mic gene were purified according to manufacturer’s recommendations, and then ligated with the pgem-teasy. thereafter, positive plasmids were transformed into the jm competent cells. following pcr screening, the positive colonies were sequenced by genscript co., ltd. sequence analysis and phylogenetic reconstruction the obtained mic gene sequences from different t. gondii strains were aligned using multiple sequence alignment program, clustal x, and sequence variation was determined among the examined t. gondii strains. the phylogenetic reconstructions based on the mic sequences from different t. gondii strains were performed by bayesian inference and maximum parsimony. bi analyses were conducted with four independent markov chains run for, metropolis coupled mcmc generations, sampling a tree every generations in mrbayes.. the first trees were omitted as burn-in and the remaining trees were used to calculate bayesian posterior probabilities. mp analysis was performed using paup* b. bootstrap support for mp tree was calculated from bootstrap replicates with random additions per replicate. pcr-rflp pcr-rflp was used to further assess whether mic gene sequence is a potential marker for genotyping t. gondii isolates. the amplified mic fragments were digested with restriction enzyme pst i for 8 h at 37 °c in the appropriate buffer, and then inactivated at 80 °c for 20 min according to the manufacturer’s instructions. the restriction fragments were analyzed by electrophoresis on 1 % agarose gel stained with goldenview™ and photographed using a gel documentation system.  sequence analysis all the positive transformants of the mic gene were approximately 4000 bp in length on 1 % agarose gel. the mic gene sequences were 4391 bp in length for strains tgcatbr and mas; and they were 4394 bp in length for strains rh, tgplh, gt pru, qho, ptg, pys, gjs, ctg and tgtoucan. their a+t content varied from to  %. alignment of the obtained mic sequences from 12 t. gondii strains revealed nucleotide polymorphisms at sequence variation positions, including variations in introns, variations in exons. in addition, a total of transversions, transitions, deletions and inserts were identified in mic gene. sequences analysis of polymorphisms in the three references genotypes revealed the existence of polymorphic restriction sites. using pcr-rflp method, digestion of the amplification products with pst i allowed the differentiation between genotype i, ii, and iii.fig. phylogram of toxoplasma gondii strains determined by analysis of the entire sequences of the mic gene. the tree was built by bayesian inference and maximum parsimony analysis. the numbers along branches show bootstrap values resulting from different analyses: bi/mp phylogenetic analysis of t. gondii strains based on mic sequences by phylogenetic reconstruction based on mic sequence data of all strains, we have obtained the phylogenetic tree. phylogenetic analysis revealed three major clusters, which are corresponding to classical genotypes respectively, and type iii are clustered more closely with type i than with other strains. atypical strains tgtoucan were phylogenetically linked to type iii, and thus the both strains gys and pys were phylogenetically linked to type ii.fig. pcr-rflp analysis of mic genomic sequences of toxoplasma gondii isolates in 1 % agarose gel using restriction endonucleases pst i. lane m represents dna size marker lanes 1– represent t. gondii type i, type ii, type iii strains respectively. refer to table  for isolate information discussion in the present study, we determined the entire genome region of the mic locus for 12 t. gondii strains among different host and geographical origins, and determined genetic diversity between toxoplasma strains based on mic gene sequences. it has been found that there were a total of variable positions distributed in the genome sequence of the mic locus among isolates, including variations in exons and variations in introns. these results suggested higher sequence variations in mic gene than that in mic mic and eif4a genes. the analyses of mic gene in both nucleotides and amino acids among 12 t. gondii strains showed high ratio of non-synonymous to synonymous polymorphisms, emphasized that mic was undergoing positive selection together with some other loci, such as gra gra and rop. with the purpose of analyzing the evolutionary relationship among the examined t. gondii isolates, the phylogenetic tree was established based on the mic sequence using bi and mp methods. phylogenetic analysis showed that t. gondii strains corresponding to classical genotypes were grouped into their respective clusters separately, which were consistent with the results from other genetic markers including gra and gra genes. moreover, the three major clonal lineages can be differentiated by digestion of mic pcr products using endonuclease pst i, which further suggested that the mic locus may be a potential new genetic marker for pcr-rflp genotyping of t. gondii strains. however, some atypical strains including tgcatbr tgtoucan, pys, gjs and mas can not be distinguished from the three major lineage types by pcr-rflp, suggesting that pcr-rflp genotyping with a single locus is not enough for the identification of non-clonal genotypes.  the present study examined sequence variation in mic and demonstrated the existence of sequence variability in mic gene among the examined t. gondii strains from different hosts and geographical localities. phylogenetic analysis and pcr-rflp genotyping of the examined t. gondii strains suggest that mic gene may provide a potential marker for population genetic studies of t. gondii isolates, and further research sampling more isolates from different hosts and geographical separations need to be done to verify this possibility. abbreviations aids, acquired immune deficiency syndrome; bi, bayesian inference; hiv, human immunodeficiency virus; mp, maximum parsimony; pcr, polymerase chain reaction; pcr-rflp, pcr-restriction fragment length polymorphism; tgmic toxoplasma gondii microneme protein 16 additional file additional file 1: figure s alignment of mic gene sequences of toxoplasma gondii strains. lower case letter “a” indicates the base changes in exons; lower case letter “b” indicates the base changes in introns. 
however, little is known of the tgmic sequence diversity among t. gondii isolates from different hosts and geographical locations. in this study, we examined sequence variation in mic gene among t. gondii isolates from different hosts and geographical regions. phylogenetic analysis and pcr-rflp of the mic locus among t. gondii isolates from different hosts and geographical regions allowed the differentiation of three major clonal lineages into their respective genotypes, suggesting that mic gene may provide a novel potential genetic marker for population genetic studies of t. gondii isolates. moreover, the three major clonal lineages can be differentiated by pcr-rflp using restriction enzyme pst i. the results of sequence alignments showed two lengths of the sequence of mic gene among all the examined 12 t. gondii strains: 4391 bp for strains tgcatbr and mas, and 4394 bp for strains rh, tgplh, gt pru, qho, ptg, pys, gjs, ctg and tgtoucan. microneme protein of t. gondii is responsible for binding to aldolase, and is associated with rhomboid cleavage and presence of trafficking signals during invasion. the entire genomic region of the mic gene was amplified and sequenced, and phylogenetic relationship was reconstructed using bayesian inference and maximum parsimony based on the mic gene sequences. their a+t content ranged from to  %. phylogenetic analysis of mic sequences showed that typical genotypes were able to be grouped into their respective genotypes.
 the zoonotic agent toxoplasma gondii is distributed world-wide, and can infect a broad range of hosts including humans. microneme protein of t. gondii is responsible for binding to aldolase, and is associated with rhomboid cleavage and presence of trafficking signals during invasion. however, little is known of the tgmic sequence diversity among t. gondii isolates from different hosts and geographical locations.  in this study, we examined sequence variation in mic gene among t. gondii isolates from different hosts and geographical regions. the entire genomic region of the mic gene was amplified and sequenced, and phylogenetic relationship was reconstructed using bayesian inference and maximum parsimony based on the mic gene sequences. the results of sequence alignments showed two lengths of the sequence of mic gene among all the examined 12 t. gondii strains: 4391 bp for strains tgcatbr and mas, and 4394 bp for strains rh, tgplh, gt pru, qho, ptg, pys, gjs, ctg and tgtoucan. their a+t content ranged from to  %. a total of variable nucleotide positions were identified, including variations in exons and variations in introns. phylogenetic analysis of mic sequences showed that typical genotypes were able to be grouped into their respective genotypes. moreover, the three major clonal lineages can be differentiated by pcr-rflp using restriction enzyme pst i.  phylogenetic analysis and pcr-rflp of the mic locus among t. gondii isolates from different hosts and geographical regions allowed the differentiation of three major clonal lineages into their respective genotypes, suggesting that mic gene may provide a novel potential genetic marker for population genetic studies of t. gondii isolates. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords toxoplasma gondiimic16sequence diversitygenetic markerpcr-rflpissue-copyright-statement© the author 2016 