the successful pathogen , toxoplasma gondii , is able to infect virtually all warm - blooded animals including birds and mammals in worldwide , obligating to the zoonotic toxoplasmosis ( 1 ) . human beings acquired the disease mainly through ingestion of tissue cysts or oocysts of t. gondii , containing in food or water ( 2 ) . t. gondii infection in healthy adults rarely causes clinical symptoms , but can lead to be fatal in immunocompromised individuals ( 24 ) . in wildlife , many animals also were infected by t. gondii . some studies reported high infection rate of t. gondii in zoo animals and wild birds . for example , murata in early 1989 just showed 29.28% ( 53/181 ) and 16.94% ( 61/179 ) of t. gondii igg in mammals and wild birds , respectively , by serological survey ( 5 ) . zhang et al . also reported there had 25% ( 4/16 ) of t. gondii antibody in primates , 69.4% ( 25/36 ) in carnivores , 27.6% ( 8/29 ) in herbivores and 11.11% ( 4/36 ) in birds in china ( 6 ) . there was 36.17% ( 51/141 ) in felids and 10.81% ( 4/37 ) in prosimians ( 7 ) . other recent reports also showed the high infection rate such as 36.1% ( 73/202 ) of wild birds captured from the wild environment ( 8) and 12.46% ( 39 /313 ) of house sparrows in china ( 9 ) . in recent years , different sources of t. gondii strains were identified using multilocus polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) method , which was established for genotyping by su and other scholars ( 1014 ) . the distribution of genetic diversity of t. gondii in wildlife animals is of great importance to understand the transmission of this parasite in the environment . some studies have identified several genetic characterizations of t. gondii isolates from cats , bats , sheep , and birds in china ( 1518 ) . however , little information concerning t. gondii isolates from zoo animals and pet birds in southeast china . the objectives of the present study were to determine the prevalence and genetically characterize t. gondii in wild animals from fuzhou zoo and pet birds farms in fujian province , southeastern china . a total of 185 animals were randomly collected from fujian province during 2012 and 2014 . the 45 heart tissue samples from were collected from zoo animals , and 140 heart tissue samples were collected from pet birds . genomic dna was extracted from heart tissue samples of wild animals and pet birds using tianamp genomic dna kit ( tiangen , beijing , china ) . in brief , 30 mg of heart tissue were treated with sodium dodecyl sulphate / proteinase k at 56 c for overnight digestion in a thermostat water bath . dna samples were prepared according to the previous study ( 19 ) and eluted into 60 ul with elution buffer . the positive genomic dna of t. gondii rh ( type i ) and pru ( type ii ) strains were kindly provided by fujian normal university and lanzhou veterinary research institute , respectively . at first , all the dna samples from tissues of wild animals and pet birds were identified using semi - nested pcr method based on t. gondii b1 gene as described previously ( 20 ) . in brief , the first round were amplified at 35 cycles with 93 c for 10 s , 57 c for 10 s and 72 c for 30 s. then the nested pcr was beginning at 93 c 10 s for denaturation , following by annealing at 62.5 c for 10 s and the 72 c 10 s for extension ( 21 ) . the pcr amplification was performed by veriti 96-well thermal cycler ( abi ) , and the products were separated by 1.5% agarose gel , visualized under ultraviolet light . the positive samples were then genotyped using multilocus pcr - rflp method as previously described by amplifications of 11 markers , including sag1 , 5 and 3-sag2 , alternative sag2 , sag3 , btub , gra6 , c22 - 8 , c29 - 2 , l358 , pk1 , apico , and cs3 ( ( 13 , ( 15 , ( 22 ) . the reaction volume ( 25 l ) contained 10 mm tris - hcl , 50 mm kcl , 1.5 mm mgcl2 , 0.3 mm each primer , 0 . 25 mm each dntp , 1.25 u golden dna polymerase ( tiangen , beijing , china ) , and 2 l t. gondii dna . all samples were incubated at 95 c for 5 min , then 35 cycles of pcr at 94 c for 30 s , 55 c for 30 s , 72 c for 40 s and 72 c for 7 min . the nested pcr was carried out with an annealing temperature at 60 c for 60 s for all the markers except apico , which was amplified at 55 c . the previous studies ( ( 9 , ( 13 , ( 14 , ( 15 ) are used to compare with the results of the genetic characterization of the chinese t. gondii in our present work . a total of 185 animals were randomly collected from fujian province during 2012 and 2014 . the 45 heart tissue samples from were collected from zoo animals , and 140 heart tissue samples were collected from pet birds . genomic dna was extracted from heart tissue samples of wild animals and pet birds using tianamp genomic dna kit ( tiangen , beijing , china ) . in brief , 30 mg of heart tissue were treated with sodium dodecyl sulphate / proteinase k at 56 c for overnight digestion in a thermostat water bath . dna samples were prepared according to the previous study ( 19 ) and eluted into 60 ul with elution buffer . the positive genomic dna of t. gondii rh ( type i ) and pru ( type ii ) strains were kindly provided by fujian normal university and lanzhou veterinary research institute , respectively . at first , all the dna samples from tissues of wild animals and pet birds were identified using semi - nested pcr method based on t. gondii b1 gene as described previously ( 20 ) . in brief , the first round were amplified at 35 cycles with 93 c for 10 s , 57 c for 10 s and 72 c for 30 s. then the nested pcr was beginning at 93 c 10 s for denaturation , following by annealing at 62.5 c for 10 s and the 72 c 10 s for extension ( 21 ) . the pcr amplification was performed by veriti 96-well thermal cycler ( abi ) , and the products were separated by 1.5% agarose gel , visualized under ultraviolet light . the positive samples were then genotyped using multilocus pcr - rflp method as previously described by amplifications of 11 markers , including sag1 , 5 and 3-sag2 , alternative sag2 , sag3 , btub , gra6 , c22 - 8 , c29 - 2 , l358 , pk1 , apico , and cs3 ( ( 13 , ( 15 , ( 22 ) . the reaction volume ( 25 l ) contained 10 mm tris - hcl , 50 mm kcl , 1.5 mm mgcl2 , 0.3 mm each primer , 0 . 25 mm each dntp , 1.25 u golden dna polymerase ( tiangen , beijing , china ) , and 2 l t. gondii dna . all samples were incubated at 95 c for 5 min , then 35 cycles of pcr at 94 c for 30 s , 55 c for 30 s , 72 c for 40 s and 72 c for 7 min . the nested pcr was carried out with an annealing temperature at 60 c for 60 s for all the markers except apico , which was amplified at 55 c . the previous studies ( ( 9 , ( 13 , ( 14 , ( 15 ) are used to compare with the results of the genetic characterization of the chinese t. gondii in our present work . of the total 185 samples , 10 samples were detected to be positive for the t. gondii b1 gene , including 7 zoo animals ( 1 black - capped , 1 wild red dog , 1 lemur , 1 zebra , 1 red panda , 1 peacock , 1 red - crowned crane , 15.56% , 7/45 ) and 3 pet birds ( 1 budgerigar , 1 black - billed magpie , 1 zebra finch , 2.14% , 3/140 , ) ( table 1 ) . the positive rate of t. gondii dna in zoo animals was significantly higher than that of pet birds in fujian , china ( p<0.05 . further genotyped the positive samples using multi - locus pcr - rflp method showed that 3 dis - tinct genotypes ( toxodb genotype # 9 , # 2 and # 10 ) of t. gondii from black - capped ( cebus apella ) , peacock ( peafowl ) and budgerigar ( melopsittacus undulatus ) respectively were identified ( table 2 ) . by using sequence - specific primers , the relative number of copies of a particular dna sequence can be determined . there are many applications of this technique in the field of diagnosis ( 2325 ) . t. gondii can be detected by semi - nested polymerase chain reaction ( pcr ) using b1 genes in pig , cat , and other wild animals ( 2629 ) . in the present study , we identified the t. gondii infection in black - capped , wild red dog , lemur , zebra , red panda , peacock , red - crowned crane , budgerigar , black - billed magpie , zebra finch in china , which indicated these animals could be served as a potential source of infection for other animals and even humans . pcr - rflp method is frequently used to identify differences between homologous dna sequences ( 13,15 ) , which have been successfully used for genotyping of t. gondii from cat , chicken , human , cougar , sheep , birds , sparrow , pig and other animals ( 2 , 9 , 13 , 1518 , 26 ) . further genotyped the positive samples using multi - locus pcr - rflp method showed that 3 distinct genotypes ( toxodb genotype # 9 , # 2 and # 10 ) of t. gondii from black - capped ( cebus apella ) , peacock ( peafowl ) and budgerigar ( melopsittacus undulatus ) respectively were identified ( table 2 ) . zoo animals and pet birds have formed a huge industry in order to prevent rare wild animals from extinction and to boost the economy . fujian province has a great advantage in convenient transportation because of its southeast coastal location in china , so the exchange of wild animals and pet birds becomes more ordinary . the results in this present study will enhance our understanding of the epidemiology and prevention of t. gondii in zoo wild animals and pet birds . one sample isolated from budgerigar belonged to toxodb genotype # 10 , supporting the previous study that toxodb genotype # 10 had been isolated from 3 sparrows in fuzhou ( 18 ) . this result indicated that toxodb genotype # 9 was predominant not only in southern , southwestern and central parts of china but also in southeast part of china ( 15 , 17 , 3032 ) . in addition , our finding support that toxodb genotype # 9 is at cs3 locus . our study is also the first report of genetic typing of t. gondii from zoo wild animals and pet birds in china . the high genetic diversity of t. gondii genotypes in fujian may cause by the high number of cats in city and other feline in mountainous areas . the positive rate of t. gondii dna was high in zoo animals ( 7/45 , 15.56% ) but very low in birds ( 3/140 , 2.14% ) in fujian , china . further genotyping analysis confirmed that the presence of t. gondii in zoo wildlife and pet birds were toxodb genotype # 10 , genotype # 9 and genotype # 2 , which suggested the integrated measures should be taken to prevent t. gondii infection in humans or other animals .
background : toxoplasmosis , a worldwide zoonotic disease , is caused by toxoplasma gondii . 
 the distribution of genetic diversity of t. gondii in wild animals is of great importance to understand the transmission of the parasite in the environment . 
 however , little is known about t. gondii prevalence in wild animals and birds in china.methods:we conducted the genetic characterization of t. gondii isolated from zoo wild animals and pet birds in fujian province , southeastern china . 
 heart tissues were collected from 45 zoo animals and 140 pet birds . 
 after identified using b1 gene , the genetic diversity of t. gondii isolates were typed at 11 genetic markers , including sag1 , 5 and 3-sag2 , alternative sag2 , sag3 , btub , gra6 , c22 - 8 , c29 - 2 , l358 , pk1 , apico , and cs3.results:seven of 45 zoo animals and 3 of 140 pet birds were positive by pcr amplification using t. gondii b1 gene specific primers . 
 of these positive isolates , 3 isolates from black - capped ( cebus apella ) , peacock ( peafowl ) and budgerigar ( melopsittacus undulatus ) were successfully genotyped at 11 genetic loci , and grouped to three distinct genotypes : toxodb genotype # 9 , # 2 and # 10 , respectively.conclusion:this is the first genotyping of t. gondii isolated from zoo wild animals and pet birds in fujian , china . 
 there is a potential risk for the transmission of this parasite through zoo wild animals and pet birds in this region .