| Avain coccidiosis is a parasitic disease caused by Eimeria spp.parasitic in chicken intestines.There are seven species of chicken coccidia.In clinical practice,chickens often harbor infection with one or more species of coccidia.Among seven species of chicken coccidia,the epidemiology,pathogenicity,immunogenicity and drug sensitivity were different from each other.Therefore,the identification of coccidia species and their epidemiological investigation are helpful to the prevention and control of chicken coccidiosis.The traditional methods for identification of coccidia species in chickens are mainly based on the biological characteristics such as oocyst morphology,zone of intestine parasitized,gross appearance of the lesion,minimum prepatent time,and so on.It not only needs to raise chickens in Eimeria-free isolation cages,but also is time-consuming and laborious.In the present study,seven pairs of species-specific primers were designed according to the sequence of ribosomal DNA internal transcribed spacer 1(ITS-1)of chicken coccidia,and the methods of single PCR and multiple PCR for identification of chicken coccidia were established.Recombinant plasmid containing coccidia ITS-1 sequence was contructed,which are used as a standard of ITS-1 DNA of coccidia.Finally,the coccidia species and epidemiology in thirty-one chicken flocks from twenty farmers in Rudong county were investigated by multiplex PCR.The method established in this study provides a technical measures for the prevention and control of chicken coccidiosis.1.Establishment of a single PCR method for identification of coccidia species of chickenAccording to the ITS-1 sequences of E.tenella,E.necatrix,E.acervulina,E.maxima,E.praecox,E.mitis and E.brunetti published in GenBank,seven pairs of species-specific primers were designed.Using these primers,a single PCR method for identification of coccidia species was established,with the genomic DNAs of chicken coccidia as template.The PCR reaction conditions were optimized,and the pecificity and sensitivity of the PCR system were evaluated,respectively.The results showed that the method amplified the special fragments with 278 bp(E.tenella),383 bp(E.necatrix),476 bp(E.acervulina),220 bp(E.maxima),390 bp(E.praecox),350 bp(E.mitis)and 311 bp(E.brunetti),respectively.The minimum detection concentration was 0.01 ng DNA,and there were no cross rections with Toxoplasma gondii,Histomonas meleagridis and Leucocytozoon spp.,indicating that the method had strong specificity and high sensitivity.2.Establishment of a multiplex PCR method for identification of coccidia species of chickenAmong the seven species of chicken coccidia,E.tenella,E.acervulina and E.maxima are the most common in clinical practice;E.tenella,E.necatrix and E.brunetti have high pathogenicity,all of them produce oocysts in the cecum,with similar oocyst morphology;E.praecox,E.mitis and E.acervulina possess similar prepatent time and oocyst morphology.The rapid identification of these coccidia species is helpful to the accurate diagnosis and control of chicken coccidiosis.For this reason,a multiplex PCR method for identification of three coccidia species was established using the above species-specific primers.The PCR reaction conditions were optimized,and the specificity and sensitivity of the PCR system were evaluated,respectively.The results showed that three pairs of primers in each multiplex PCR could amplify the special fragments.The minimum detection concentration was 0.01 ng DNA.There were no cross rections with Toxoplasma gondii,Histomonas meleagridis and Leucocytozoon spp.,indicating that the multiplex PCR method had strong specificity and high sensitivity.3.Construction of recombinant plasmid containing ITS-1 sequence of chicken coccidia and its application in PCR detectionThe products purified from the single PCR were ligated into pGEM-TEasy vector and transferred into E.coli DH5a cells.The recombinant plasmid containing ITS-1 sequence was obtained by blue-white spot screening and restriction enzyme digestion.The inserted fragments were sequenced.The sequences of ITS-1 were analyzed by MegAgin7.1.0 software,and the phylogenetic tree was constructed by Neighbor-joining method.The recombinant plasmid DNA containing coccidia ITS-1 were used as template for PCR amplification,and DNA produced from oocyst of seven coccidia species as positive control.The results showed that the ITS-1 fragments of E.tenella,E.necatrix,E.maxima,E.acervulina,E.praecox,E.mitis and E.brunetti were 278 bp,383 bp,220 bp,476 bp,390 bp,350 bp,311 bp,respectively.The stains used in this study belong to a clade with the corresponding species in GenBank.The recombinant plasmid DNA could be used as a template for PCR.4.Preliminary application of multiplex PCR method in epidemiological investigation of chickencoccidiosisThe species of chicken coccida in 31 chicken flocks from 20 farms in Rudong County were investigated by saturated saline floating method and multiple PCR method.The results showed that 65%(13/20)of the chicken farms and 64.5%(20/31)of the flocks were positive for oocysts with saturated saline floating test,and 75%(15/20)of the chicken farms and 70.9%(22/31)of the flocks were positive for oocysts by multiple PCR,respectively.A total of seven species of coccidia were detected.The positive rates of E.tenella,E.necatrix,E.maxima,E.acervulina,E.praecox,E.mitis and E.brunetti were 64.5%(20/31),38.7%(12/31),35.5%(11/31),45.2%(14/31),19.4%(6/31),19.4%(6/31)and 12.9%(4/31),respectively.The dominant species was E.tenella,and the positive rate of E.brunetti was the lowest;all the positive samples(100%)were mixed infection of more than two coccidia species,in which the rate of mixed infection of two coccidia species were 36.3%(8/22).The positive rates of coccidia infection was higher in captive breeding(80.0%)than in cage rearing(33.3%).The result provided a basis for effective control of chicken coccidiosis in Rudong County. |
