Survey Of Epidemiology And Study On Molecular Characters Of Encephalitozoon Cuniculi In Rabbits In Henan Province

Encephalitozoon cuniculi is an important and zoonotic pathogeny infecting nearly all of the animals including human.It is possible to spread by water and the main symptom is neurological symptoms in rabbits and human eye diseases.It is reported in China the infection rate of Encephalitozoon cuniculi in rabbits is higher using serological methods, up to 76%. foreign-related infection rate is also higher in reports , up to 85%. In the world,the reports of investigate the prevalence rate of Encephalitozoon cuniculi in rabbits using molecular biological methods is less.From September 2009 to December 2010, in order to report the prevalence rate of Encephalitozoon cuniculi in rabbits in Henan province by using the Chromotrope 2R staining methods and PCR to investigate,including nine large-scale rabbit farms,one pet market and one rabbit slaughter from 7 areas and study on molecular characteristics of positive samples.1. In order to understand the infected condition of Encephalitozoon cuniculi in some areas of Henan Provice, samples of fresh stool obtained from the rabbit farms, which were detected for spores of Encephalitozoon cuniculi by microscope of Chromotrope 2R staining method between September 2009 and September 2010. The areas contained 9 large-scale rabbit farms(including two pet rabbit farm) and one pet market in Zhengzhou,Zhoukou,Jiaozuo,Puyang,Zhumadian and Nanyang.After staining the spores were stained red with refraction while bacteria and fecal residue dyed green.The spores were oval or ellipse with 1-3μm. Not all the spores had been dyed homogeneous red. Some spore wall were darker colored and the center of spores were light color or pale. Many spores also showed a typical band structure, that was, diagonal or vertical line of the belt-like surrounding the spores. 305 pet rabbits and 1027 rabbit stool samples were collected.No positive were found in pet rabbits and 38 of 1027 fecal specimens were diagnosed as Encephalitozoon cuniculi, the Encephalitozoon cuniculi infection rate was 3.7%.And the infection rate is different among different sampling points,different ages and different breeds in rabbits.The infection rate in Puyang and Zhoukou cities is 4.7%,8.1%, respectively.No positive was found in Jiaozuo and Nanyang cities. The infection rate of Encephalitozoon cuniculi was 2.0%,5.1% and 4.2% in 1-3 months old,3-7 months old and over 7 months of rabbits,respectively. The infection rate of Encephalitozoon cuniculi was 0.5%,6.0% and 3.0% in angora rabbit,rabbit and rex rabbit, respectively. 2.To further determine the infection condition of Encephalitozoon cuniculi and to provide a reference for the clinical detection, samples of 72( each sample contained stool and urine samples from one rabbit )were collected from an experimental rabbit farm and a rabbit slaughter in Zhengzhou and Jiyuan of Henan Province.Use nest-PCR to amplify 16srRNA gene of Encephalitozoon cuniculi, the results showed that no positive in stool samples and 5 positive samples were amplified from urine samples,and the infection rate was 5.6%.The sequence length of products were 549bp.The results showed that this may not be excreted through feces.The spores were found in mixed samples between feces and urine samples,suggesting that the rection rate was detected by mixed samples in clinical.3. To study on molecular characteristics of Encephalitozoon cuniculi,5 positive samples amplified by nested PCR were sequenced. Then, Blast methods was used to search homological sequences in NCBI, after that, homological sequences were alignmented. Phylogenetic tree and homological analysis were made by some biological softwares such as Clustal X 1.83,Phylip3.65,TreeView and DNAstar 4.0.The results showed that all of the 5 samples were Encephalitozoon cuniculi and the individual bases were difference between 5 sequences.The homology between one sequence in this syudy and another sequence in Blast was up to 100%.The Encephalitozoon cuniculi of rabbits is one of the important zoonosis, so it is fairly important significance of public health to research the Encephalitozoon cuniculi of rabbits.Using Chromotrope 2R staining method and PCR method to study the infection condition of Encephalitozoon cuniculi in rabbits have not been reported in China.Therefore, The results of tihs study will provide a reference for research the Encephalitozoon cuniculi of rabbit such as molecular epidemiology, biologic characters and the relationship of germ line evolution between the isolates.