Epidemiology Investigation On Some Intestinal Protozoans From Free-Range Chickens In Anhui Province

Intestinal protozoa are common parasites in poultry.Intestinal protozoa parasites not only affect the growth and development of poultry,but also cause death of poultry,causing huge economic losses to poultry industry.At the same time,some protozoa infected with poultry are zoonotic or have the potential of zoonosis.Poultry can become the source of infection of these protozoa,which is of great significance in public health.In recent years,the chicken industry in Anhui Province has developed rapidly,especially the number of free-range chickens has increased rapidly,but the prevention and control of parasitic diseases in free-range chickens has lagged behind seriously,especially the epidemic situation of many intestinal parasites is still blank.In view of this,this study intends to investigate the molecular epidemiology of T.gallinarum,Cryptosporidium,E.bieneusi,G.duodenalis,Blastocystis sp.,H.meleagridis in scattered chicken flocks in Anhui Province,in order to provide reference for the prevention and control of parasitic diseases in scattered chickens in Anhui Province and the evaluation of the role of scattered chickens in the transmission process of zoonotic protozoa and its public health significance.To determine the occurrence of T.gallinarum in free-range chickens in Anhui Province,we collected fresh fecal samples from 829 chicken located on Mingguang City of Chuzhou,Longqiao District of Suzhou,Fengtai City of Huainan,Ningguo City of Xuan Cheng,Taihe County of Fuyang City and Jinan District of Luan City.After genomic DNA was extracted from all samples,T.gallinarum SSU rRNA gene-specific PCR method was used.The results showed that the infection rate of T.gallinarum was 2.1%(17/829)in free-range chicken flocks in Anhui Province.The infection rate of T.gallinarum was 4.6%(5/108)in Suzhou,4.0%(2/50)in Huainan and 1.0%(1/99)in Chuzhou.No T.gallinarum was detected in other areas.Sequence analysis of SSU rRNA showed that all 16 sequences were T.gallinarum.Three representative T.gallinarum SSU rRNA genes were obtained.Phylogenetic tree showed that all T.gallinarum strains clustered in one branch.The three sequences obtained in this study were located in different clusters,suggesting that the chicken T.gallinarum SSU rRNA gene obtained in this study had certain genetic diversity.To determine the prevalence and molecular characterization of Cryptosporidium isolates from free-range chicken in Anhui province,all specimens DNA from free-range chicken were detected using a PCR targeting 18S rRNA gene of Cryptosporidium.At the same time,the samples identified as C.meleagridis were identified as gp60 gene.To identify Cryptosporidium genotype by amplification and analysis.The results showed that the infection rate of Cryptosporidium was 1.3%(11/829)in free-range chicken flocks in Anhui Province.The infection rate of Crytosporidium was 4.6%(5/108)in Suzhou,2.0%(1/50)in Huainan,2.6%(3/116)in Xuancheng 1 and 2.1%(2/96)in Xuancheng 2.No positive samples of Cryptosporidium were detected in other areas.Sequence analysis showed that four Cryptosporidium species were detected in this study.C.baileyi,C.meleagridis,C.galli and Cryptosporidium sp.(C.bovis-like),of which C.baileyi is the dominant species.The analysis of gp60 gene showed that the two C.meleagridis genotypes obtained in this study were all ⅢbA26G1R1b.To determine the prevalence and molecular characterization of E.bieneusi isolates from free-range chicken in Anhui province,all specimens DNA from free-range chicken were detected using a PCR targeting ITS gene of E.bieneusi.The results showed that the total infection rate of E.bieneusi was 1.6%(13/829)in free-range chickens in Anhui Province.ITS gene sequence analysis showed that six E.bieneusi genotypes were obtained in this study,including one known genotype D and five new genotypes ABC1,AHC2,AHC3,AHC4 and AHC5.The construction of evolutionary tree showed that all genotypes obtained in this study belonged to Group 1.To determine the prevalence and molecular characterization of G.duodenalis isolates from free-range chicken in Anhui province,all specimens DNA from free-range chicken were detected using a PCR targeting SSU rRNA gene of G.duodenalis.The results showed that G.duodenalis was negative in free-range chicken,suggesting that there was no G.duodenalis pollute in the environment around the free-range chicken farms in Anhui Province.To determine the prevalence and molecular characterization of Blastocystis sp.isolates from free-range chickens in Anhui province,all specimens DNA from free-range chickens were detected by PCR based on the "barcode region" of the small subunit ribosomal RNA(18S rRNA)gene of Blastocystis sp.The sequenced obtained were submitted to Blastocystis sp.18s database to determine its genotype and 18s allele.The occurrence of Blastocystis sp.was 1.5%(12/829),of which Chuzhou was 1.0%(1/829),Suzhou was 6.5%(7/829),Xuancheng was 3.5%(4/829),and no positive samples of Blastocystis sp.were detected in other places.Sequence analysis showed that the sheep carried ST6 and ST7.The 18s allele analysis showed that genotype ST6 have only one new allele as Novel allele 1,ST7 have one known allele 96 and two new alleles Novel allele 2 and Novel allele 3.To determine the prevalence and molecular characterization of H.meleagridis isolates from free-range chicken in Anhui province,all specimens DNA from free-range chicken were detected using a nested-PCR targeting 18S rRNA gene of H.meleagridis.The results showed that the infection rate of H.meleagridis was 0.2%(2/829)in Nest’s PCR reaction,and the two positive samples were all distributed in Luan,the infection rate was 0.8%(2/239).