however , a trans - placental and respiratory route of infection has been reported in rabbits , too ( 4 ) . rabbits suffering from encephalitozoonosis show various clinical signs , and most infections are initially asymptomatic until sudden death . many rabbits infected with e. cuniculi subsequently develop renal failure , eye lesions , and neurological signs ( 5 , 6 ) . encephalitozoon cuniculi , together with other microsporidia species , has emerged as an opportunistic infection in immunocompromised patients , i.e. persons suffering from aids ( 79 ) . in addition , viable e. cuniculi was isolated from aids patients in many parts of the world ( 1012 ) . a few is known about its prevalence in rabbits and human from east asia . in japan , high prevalence of e. cuniculi in not only diseased but also healthy rabbits and human was demonstrated ( 13 , 14 ) . until now , nothing is known about the prevalence of this parasite in rabbits and humans in china , this study is the first survey to evaluate the prevalence of anti - e . cuniculi antibodies in rabbits and human in china by using the enzyme - linked immunosorbent assay ( elisa ) , which might facilitate the development of rational strategies for disease control and management . overall , 300 serum samples each from clinically healthy rabbits and human were collected from january to september 2013 . the collection of human and animal serum samples was approved by the ethical committee of the college of animal science , henan institute of science and technology , china ( approved no . the samples were collected from three regions in china including northeast china ( jilin province ) and southwest china ( sichuan province , chongqing municipality ) . the reason for choosing these locations was that these three regions are the major rabbit producing as well as consuming provinces . due to the different climate and geographical environment , the rex rabbit , japanese white rabbit and new zealand rabbit were the main breed , respectively . the e. cuniculi used in the present study was a rabbit strain isolate and well conserved in liquid nitrogen in henan higher education engineering technology research center for animal diseases control and residues supervision , henan institute of science and technology , china . e. cuniculi spores were produced on the rk 13 cell line in minimal essential medium with antibiotics ( 10 u penicillin / ml ; 0.1 mg streptomycin/ ml and 0.25 mg amphotericine / ml ) and 5% fetal bovine serum . spores were purified by density gradient centrifugation with percoll ( sigma - aldrich , st . louis , usa ) using a standard procedure ( visvesvara et al . , 1999 ) . following three cycles of freezing / thawing , spores of e. cuniculi were mixed with solid glass beads ( 400600 micrometer diameter , jencons scientific limited , west sussex , uk ) and sonicated ( 10 min , 60 w ) . the number of spore pre and post sonication was counted in a hemocytometer to ensure at least 95% spore disruption in the homogenate . the protein concentration of the supernatant was determined with a bca protein assay kit ( bio - rad , hercules , ca , usa ) . the soluble antigen solution was stored at 20c until use . an indirect enzyme - linked immunosorbent assay ( elisa ) corning , n.y . ) was coated with 1 g soluble e. cuniculi antigen diluted in 100 l carbonate bicarbonate buffer ( 100 mm , ph 9.6 ) and incubated at 4c for 3 days . the well surface was blocked with 5% bsa in 0.05% pbst ( pbs containing 0.05% v / v tween-20 ) at 37c for 2 h , followed by washing five times in 0.05% pbst . the conjugate was horseradish peroxidase ( hrp ) conjugated goat anti - rabbit igg or goat anti - human igg ( southern biotechnology , birmingham , alabama , usa ) , and diluted 1:6,000 in pbs . substrate tetramethylbenzidine ( tmb , tiangen biotech , beijing , china ) was then added and incubated at room temperature for 20 min . 100 l sodium acid ( 100 ng / ml ) were added to stop the colour reaction . the serum with absorbance at least 2.1-fold higher than that of the negative control serum was considered positive . statistical analyses of e. cuniculi prevalence in different administrative regions and breed groups were performed by x 
 -test . correlations between e. cuniculi infection in humans and rabbits were tested with pearson s rank correlation coefficient . statistical analysis was performed using spss 16 software for windows ( spss inc , chicago , illinois , usa ) . overall , 300 serum samples each from clinically healthy rabbits and human were collected from january to september 2013 . the collection of human and animal serum samples was approved by the ethical committee of the college of animal science , henan institute of science and technology , china ( approved no . the samples were collected from three regions in china including northeast china ( jilin province ) and southwest china ( sichuan province , chongqing municipality ) . the reason for choosing these locations was that these three regions are the major rabbit producing as well as consuming provinces . due to the different climate and geographical environment , the rex rabbit , japanese white rabbit and new zealand rabbit were the main breed , respectively . the e. cuniculi used in the present study was a rabbit strain isolate and well conserved in liquid nitrogen in henan higher education engineering technology research center for animal diseases control and residues supervision , henan institute of science and technology , china . e. cuniculi spores were produced on the rk 13 cell line in minimal essential medium with antibiotics ( 10 u penicillin / ml ; 0.1 mg streptomycin/ ml and 0.25 mg amphotericine / ml ) and 5% fetal bovine serum . spores were purified by density gradient centrifugation with percoll ( sigma - aldrich , st . louis , usa ) using a standard procedure ( visvesvara et al . , 1999 ) . following three cycles of freezing / thawing , spores of e. cuniculi were mixed with solid glass beads ( 400600 micrometer diameter , jencons scientific limited , west sussex , uk ) and sonicated ( 10 min , 60 w ) . the number of spore pre and post sonication was counted in a hemocytometer to ensure at least 95% spore disruption in the homogenate . the protein concentration of the supernatant was determined with a bca protein assay kit ( bio - rad , hercules , ca , usa ) . an indirect enzyme - linked immunosorbent assay ( elisa ) was used to detect e. cuniculi antibodies . corning , n.y . ) was coated with 1 g soluble e. cuniculi antigen diluted in 100 l carbonate bicarbonate buffer ( 100 mm , ph 9.6 ) and incubated at 4c for 3 days . the well surface was blocked with 5% bsa in 0.05% pbst ( pbs containing 0.05% v / v tween-20 ) at 37c for 2 h , followed by washing five times in 0.05% pbst . the conjugate was horseradish peroxidase ( hrp ) conjugated goat anti - rabbit igg or goat anti - human igg ( southern biotechnology , birmingham , alabama , usa ) , and diluted 1:6,000 in pbs . substrate tetramethylbenzidine ( tmb , tiangen biotech , beijing , china ) was then added and incubated at room temperature for 20 min . 100 l sodium acid ( 100 ng / ml ) were added to stop the colour reaction . the serum with absorbance at least 2.1-fold higher than that of the negative control serum was considered positive . statistical analyses of e. cuniculi prevalence in different administrative regions and breed groups were performed by x 
 -test . correlations between e. cuniculi infection in humans and rabbits were tested with pearson s rank correlation coefficient . statistical analysis was performed using spss 16 software for windows ( spss inc , chicago , illinois , usa ) . in the present study , 600 serum samples ( 300 each from rabbit and human ) were collected and analyzed by elisa to detect the antibodies against the e. cuniculi . an overall seroprevalence of 18.67% ( 56/300 ) was recorded in the rabbits . in case of breed wise seroprevalence of e. cuniculi , the rex rabbit from jilin province was found more infected ( 41.00% , p<0.01 ) as compared with japanese white rabbit from sichuan province ( 9.00% ) and new zealand rabbit from chongqing municipality ( 6.00% ) ( table 1 ) . seroprevalence of 

 e. cuniculi 

 infection in rabbits in china values bearing a different superscript letter ( a , b ) within a column differ significantly from one another ( 

 p 

 < 0.05 
 ) twenty - nine ( 9.67% ) out of 300 human serum samples were found positive for anti - e . cuniculi antibodies in jilin province ( 18.00% ) followed by sichuan province and chongqing municipality with the prevalence rate of 6.00% and 5.00% respectively . the seroprevalence in males was 10.00% ( 15/150 ) and in females was 9.33% ( 14/150 ) ( table 2 ) . thus , the gender was not significantly associated with e. cuniculi infection in this study ( p>0.05 ) . seroprevalence of 

 e. cuniculi 

 infection in humans in china values bearing a different superscript letter ( a , b ) within a column differ significantly from one another ( 

 p 

 < 0.05 
 ) there was a direct correlation between e. cuniculi infection in humans and e. cuniculi infection in rabbits ( r = 1.000 , p<0.01 ) . many serological surveys of e. cuniculi infections in different animals have been conducted using diverse methods in different areas in the world ( 1720 ) . however , very few studies have investigated the distribution of e. cuniculi infections in china . meng et al reported that 16.7% of the fox serum samples collected in liaoning province were positive , while 2% of the dog serum samples collected in beijing , shanghai , and hunan were positive according to the elisa using rswp1(17 ) . until now , nothing is known about the prevalence of this parasite in rabbits and humans in china . the findings of the present study highlighted an alarming situation that the parasite is widespread in clinically healthy rabbits and human in china . the present survey showed that the overall seropositivity for e. cuniculi infection in domestic rabbits was 18.67% , which was similar to that observed in nigeria ( 16.5% ) ( 21 ) and egypt ( 15.0% ) ( 22 ) but lower than that in the united kingdom ( 52.0% ) ( 23 ) and italy ( 31.675.4% ) ( 2426 ) . these differences may result from the different serological tests , rabbit populations , or climatic factors or to some combination of these conditions . in particular , the rex rabbit were showed to be associated to a higher frequency of infection respect to japanese white rabbit and new zealand rabbit ( p<0.01 ) . it is noteworthy to mention that also lonardi et al reported a higher seroprevalence in rabbits of the x breed than y breed and z breed ( 26 ) . these results indicate that there may be a potential association between the genetic line and the seropositivity against e. cuniculi . the overall seropositivity for e. cuniculi infection in humans was 9.67% , which was similar to that observed in the czech republic ( 1016% ) ( 27 ) but lower than that in the slovakia ( 26.4% ) ( 28 ) . in addition , viable e. cuniculi was isolated from aids patients in many parts of the world ( 1012 ) . the findings of the e. cuniculi from human cases from three regions of the china revealed zoonotic sources of infection involving rabbits to be major route of transmission in humans . in this study , the gender of human was not significantly associated with e. cuniculi infection ( p>0.05 ) , which was consistent with other reports ( 23 , 26 ) . among three provinces / municipalities , the highest prevalence of e. cuniculi infection in domestic rabbits and humans e. cuniculi spores are often ingested or inhaled through food or soil contaminated with infected urine . the spores are environmentally resistant and can survive on the ground for several weeks or months ( 22 ) . in addition , it is likely that the majority of rabbits are infected at a very early age from their mother . these results indicated that the environment of the jilin province had been seriously contaminated with infected e.cuniculi spores . in addition , close contact between owners and their rabbits could lead to an increased exposure to e. cuniculi . therefore , it is necessary for rabbit raisers , public health authorities to be aware of this problem in these regions . comprehensive practical control approaches and measures , such as the improvement of feeding conditions and management of rabbit , serologic screening of breeding stock with elimination of e. cuniculi positive reactors , should be executed . persons should avoid contact with the urine of infected or healthy rabbits , and always use good personal hygiene when washing cages and handling the rabbits . an overall seroprevalence of 18.67% ( 56/300 ) was recorded in the rabbits . in case of breed wise seroprevalence of e. cuniculi , the rex rabbit from jilin province was found more infected ( 41.00% , p<0.01 ) as compared with japanese white rabbit from sichuan province ( 9.00% ) and new zealand rabbit from chongqing municipality ( 6.00% ) ( table 1 ) . seroprevalence of 

 e. cuniculi 

 infection in rabbits in china values bearing a different superscript letter ( a , b ) within a column differ significantly from one another ( 

 p 

 < 0.05 
 ) twenty - nine ( 9.67% ) out of 300 human serum samples were found positive for anti - e . cuniculi antibodies . the findings of the present study revealed highest prevalence of anti - e . cuniculi antibodies in jilin province ( 18.00% ) followed by sichuan province and chongqing municipality with the prevalence rate of 6.00% and 5.00% respectively . the seroprevalence in males was 10.00% ( 15/150 ) and in females was 9.33% ( 14/150 ) ( table 2 ) . thus , the gender was not significantly associated with e. cuniculi infection in this study ( p>0.05 ) . seroprevalence of 

 e. cuniculi 

 infection in humans in china values bearing a different superscript letter ( a , b ) within a column differ significantly from one another ( 

 p 

 < 0.05 
 ) there was a direct correlation between e. cuniculi infection in humans and e. cuniculi infection in rabbits ( r = 1.000 , p<0.01 ) . many serological surveys of e. cuniculi infections in different animals have been conducted using diverse methods in different areas in the world ( 1720 ) . however , very few studies have investigated the distribution of e. cuniculi infections in china . meng et al reported that 16.7% of the fox serum samples collected in liaoning province were positive , while 2% of the dog serum samples collected in beijing , shanghai , and hunan were positive according to the elisa using rswp1(17 ) . until now , nothing is known about the prevalence of this parasite in rabbits and humans in china . the findings of the present study highlighted an alarming situation that the parasite is widespread in clinically healthy rabbits and human in china . the present survey showed that the overall seropositivity for e. cuniculi infection in domestic rabbits was 18.67% , which was similar to that observed in nigeria ( 16.5% ) ( 21 ) and egypt ( 15.0% ) ( 22 ) but lower than that in the united kingdom ( 52.0% ) ( 23 ) and italy ( 31.675.4% ) ( 2426 ) . these differences may result from the different serological tests , rabbit populations , or climatic factors or to some combination of these conditions . interestingly , the breed seemed to influence the distribution of e. cuniculi seropositivity . in particular , the rex rabbit were showed to be associated to a higher frequency of infection respect to japanese white rabbit and new zealand rabbit ( p<0.01 ) . it is noteworthy to mention that also lonardi et al reported a higher seroprevalence in rabbits of the x breed than y breed and z breed ( 26 ) . these results indicate that there may be a potential association between the genetic line and the seropositivity against e. cuniculi . the overall seropositivity for e. cuniculi infection in humans was 9.67% , which was similar to that observed in the czech republic ( 1016% ) ( 27 ) but lower than that in the slovakia ( 26.4% ) ( 28 ) . in addition , viable e. cuniculi was isolated from aids patients in many parts of the world ( 1012 ) . the findings of the e. cuniculi from human cases from three regions of the china revealed zoonotic sources of infection involving rabbits to be major route of transmission in humans . in this study , the gender of human was not significantly associated with e. cuniculi infection ( p>0.05 ) , which was consistent with other reports ( 23 , 26 ) . among three provinces / municipalities , the highest prevalence of e. cuniculi infection in domestic rabbits and humans e. cuniculi spores are often ingested or inhaled through food or soil contaminated with infected urine . the spores are environmentally resistant and can survive on the ground for several weeks or months ( 22 ) . in addition , it is likely that the majority of rabbits are infected at a very early age from their mother . these results indicated that the environment of the jilin province had been seriously contaminated with infected e.cuniculi spores . in addition , close contact between owners and their rabbits could lead to an increased exposure to e. cuniculi . therefore , it is necessary for rabbit raisers , public health authorities to be aware of this problem in these regions . comprehensive practical control approaches and measures , such as the improvement of feeding conditions and management of rabbit , serologic screening of breeding stock with elimination of e. cuniculi positive reactors , should be executed . persons should avoid contact with the urine of infected or healthy rabbits , and always use good personal hygiene when washing cages and handling the rabbits . therefore , the fields of veterinary and human medicine in china should be aware about this zoonotic issue and precautionary measure should be taken to avoid the spread of encephalitozoonosis .
background : encephalitozoon cuniculi is a microsporidian parasite commonly found in rabbits that can infect humans , causing encephalitozoonosis . our objective in this study was to evaluate the seroprevalence of this parasite in rabbits and humans in chinamethods : overall , 300 serum samples each from clinically healthy rabbit and human were collected from three regions of china ( sichuan province , chongqing municipality and jilin province ) from january to september 2013 and tested for anti - e . 
 cuniculi antibodies using an elisa.results:an overall seroprevalence of e. cuniculi was recorded as 56/300 ( 18.76% ) and 29/300 ( 9.76% ) in rabbit and human sera , respectively . 
 the seropositivity of rabbit samples collected from jilin province was 41% , which was significantly higher ( p<0.01 ) than sichuan province ( 9% ) and chongqing municipality ( 6% ) . 
 three breeds of rabbit were used in the present study and antibody detection in rex rabbit was significantly ( p<0.01 ) higher than japanese white and new zealand rabbit . in human , 
 jilin province was more prevalent ( 18% ) followed by sichuan province ( 6% ) and chongqing municipality ( 5%).conclusions : the e. cuniculi was present and widespread among healthy rabbits and humans in china